Neutralizing antibodies and methods of use thereof

ABSTRACT

This invention provides monoclonal antibodies that recognize the Toll-like Receptor 4/MD-2 receptor complex, and monoclonal antibodies that recognize the TLR4/MD2 complex as well as TLR4 when not complexed with MD-2. The invention further provides methods of using the humanized monoclonal antibodies as therapeutics. This invention also provides soluble chimeric proteins, methods of expressing and purifying soluble chimeric proteins, and methods of using soluble chimeric proteins as therapeutics, in screening assays and in the production of antibodies.

RELATED APPLICATION

This application is a continuation-in-part of U.S. patent applicationSer. No. 11/009,939, filed Dec. 10, 2004, which claims the benefit ofU.S. Provisional Application No. 60/528,812, filed Dec. 10, 2003; ofU.S. Provisional Application No. 60/528,811, filed Dec. 10, 2003; and ofU.S. Provisional Application No. 60/528,962, filed Dec. 10, 2003; eachof which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention relates generally to the generation of neutralizingmonoclonal antibodies, e.g., humanized monoclonal antibodies, thatrecognize the Toll-like Receptor 4/MD-2 receptor complex, to monoclonalantibodies, e.g., humanized monoclonal antibodies, that recognize boththe Toll-like Receptor 4/MD-2 receptor complex and Toll-like Receptor 4when not complexed with MD-2, and to methods of using the monoclonalantibodies as therapeutics.

BACKGROUND OF THE INVENTION

Toll receptors, first discovered in Drosophila, are type I transmembraneprotein having leucine-rich repeats (LRRs) in the extracellular portionof the protein, and one or two cysteine-rich domains. The mammalianhomologs of the Drosophila Toll receptors are known as “Toll-likereceptors” (TLRs). TLRs play a role in innate immunity by recognizingmicrobial particles and activating immune cells against the source ofthese microbial particles.

Currently, ten types of Toll-like receptors have been identified inhumans, TLRs 1-10. These TLRs are characterized by the homology of theirintracellular domains to that of the IL-1 receptor, and by the presenceof extracellular leucine-rich repeats. The different types of TLRs areactivated by different types of microbial particles. For example, TLR4is primarily activated by lipopolysaccharide (LPS), while TLR2 isactivated by lipoteichoic (LTA), lipoarabinomannan (LAM); lipoprotein(BLP), and peptideglycans (PGN). Toll receptor homologs, such as RP105,have also been identified.

Myeloid differentiation protein-2 (MD-2), a TLR4 accessory protein, hasbeen identified and characterized. This protein has been found tointeract directly with TLR4, and MD-2 has the ability to enablepost-translational modifications of TLR4, as well as facilitate itstransport to the cell surface. TLR4 and MD-2 form a complex on the cellsurface.

Lipopolysaccharide (LPS), a component of gram-negative bacteria, is amicrobial particle capable of strongly activating the innate immunesystem. LPS delivers signals to immune cells via its multi-chainreceptor, comprising the TLR4/MD-2 complex as the principle signalingcomponent.

Accordingly, there exists a need for methods and compositions thatmodulate signaling that is mediated by the TLR4/MD-2 complex.

SUMMARY OF THE INVENTION

The invention provides monoclonal antibodies recognizing the TLR4/MD-2receptor expressed on the cell surface. The antibodies are capable ofblocking, e.g., neutralizing, LPS-induced pro-inflammatory cytokineproduction. The monoclonal antibody is, e.g., a humanized antibody.Antibodies of the invention include antibodies that bind the humanTLR4/MD-2 receptor complex and also bind TLR4 independently of thepresence of MD-2. Antibodies of the invention also include antibodiesthat bind the TLR4 portion of the human TLR4/MD-2 receptor complex, butbinding is entirely dependent on the presence of MD-2. In addition,antibodies of the invention include antibodies that bind the humanTLR4/MD-2 receptor complex and also bind MD-2 but only in the presenceof TLR4.

Exemplary antibodies of the invention include, for example, the 18H10antibody, the 16G7 antibody, the 15C1 antibody and the 7E3 antibody.These antibodies show specificity for the human TLR4/MD-2 receptorcomplex, and they have been shown to inhibit receptor activation andsubsequent intracellular signaling via LPS. These antibodies havedistinct specificities. For example, 15C1 binds TLR4 independently ofthe presence of MD-2, 7E3 binds to TLR4, but binding is dependent on thepresence of MD-2, and 18H10 binds to MD-2, but requires the presence ofTLR4, as the MAb does not bind soluble forms of MD-2.

As used herein, the terms “16G7”, “mu16G7”, “7E3”, “mu7E3”, “15C1”,“mu15C1”, “18H10” or “mu18H10” refer to the murine monoclonal antibody,and the terms “hu7E3”, “hu15C1”, or “hu18H10” refer to the humanizedmonoclonal antibody.

The murine monoclonal antibodies of the invention contain a heavy chainvariable region having the amino acid sequence of SEQ ID NOS: 2, 12, 22or 32 and a light chain variable region having the amino acid sequenceof SEQ ID NOS: 7, 17, 27 or 37. The three heavy chain CDRs include anamino acid sequence at least 90%, 92%, 95%, 97% 98%, 99% or moreidentical a sequence selected from the group consisting of DSYIH (SEQ IDNO:3); WTDPENVNSIYDPRFQG (SEQ ID NO:4), GYNGVYYAMDY (SEQ ID NO:5); DYWIE(SEQ ID NO:13); EILPGSGSTNYNEDFKD (SEQ ID NO:14); EERAYYFGY (SEQ IDNO:15); GGYSWH (SEQ ID NO:23); YIHYSGYTDFNPSLKT (SEQ ID NO:24);KDPSDGFPY (SEQ ID NO:25); TYNIGVG (SEQ ID NO:33); HIWWNDNIYYNTVLKS (SEQID NO:34); and MAEGRYDAMDY (SEQ ID NO:35) and a light chain with threeCDR that include an amino acid sequence at least 90%, 92%, 95%, 97% 98%,99% or more identical to a sequence selected from the group consistingof the amino acid sequence of SASSSVIYMH (SEQ ID NO:8); RTYNLAS (SEQ IDNO:9); HQWSSFPYT (SEQ ID NO:10); RSSQSLENSNGNTYLN (SEQ ID NO:18);RVSNRFS (SEQ ID NO:19); LQVTHVPPT (SEQ ID NO:20); RASQSISDHLH (SEQ IDNO:28); YASHAIS (SEQ ID NO:29); QNGHSFPLT (SEQ ID NO:30); RASQDITNYLN(SEQ ID NO:38); YTSKLHS (SEQ ID NO:39); and QQGNTFPWT (SEQ ID NO:40).The antibody binds to the TLR4/MD-2 complex, to TLR4 when not complexedwith MD-2, or to both.

The humanized antibodies of the invention contain a heavy chain variableregion having the amino acid sequence of SEQ ID NOS: 45, 46, 49, 51 and52. The humanized antibodies of the invention contain a light chainvariable region having the amino acid sequence of SEQ ID NOS: 47, 48 50,and 53. The three heavy chain CDRs include an amino acid sequence atleast 90%, 92%, 95%, 97% 98%, 99% or more identical a sequence selectedfrom the group consisting of GGYSWH (SEQ ID NO:23); YIHYSGYTDFNPSLKT(SEQ ID NO:24); KDPSDGFPY (SEQ ID NO:25); DSYIH (SEQ ID NO:3);WTDPENVNSIYDPRFQG (SEQ ID NO:4), GYNGVYYAMDY (SEQ ID NO:5); TYNIGVG (SEQID NO:33); HIWWNDNIYYNTVLKS (SEQ ID NO:34); and MAEGRYDAMDY (SEQ IDNO:35). The three light chain CDRs include an amino acid sequence atleast 90%, 92%, 95%, 97% 98%, 99% or more identical to a sequenceselected from the group consisting of the amino acid sequence ofRASQSISDHLH (SEQ ID NO:28); YASHAIS (SEQ ID NO:29); QNGHSFPLT (SEQ IDNO:30); SASSSVIYMH (SEQ ID NO:8); RTYNLAS (SEQ ID NO:9); HQWSSFPYT (SEQID NO:10); RASQDITNYLN (SEQ ID NO:38); YTSKLHS (SEQ ID NO:39); andQQGNTFPWT (SEQ ID NO:40). The antibody binds to the TLR4/MD-2 complex,to TLR4 when not complexed with MD-2, or to both.

Antibodies of the invention immunospecifically bind a TLR4/MD-2 complex,wherein the antibody binds to an epitope that includes one or more aminoacid residues on human TLR4 between residues 289 and 375 of SEQ IDNO:54. For example, the antibody specifically binds to an epitope thatincludes residues selected from the group consisting of at leastresidues 293 through 295 of SEQ ID NO:54; at least residues 296 and 297of SEQ ID NO:54; at least residues 319 through 321 of SEQ ID NO:54; atleast residues 328 and 329 of SEQ ID NO:54; at least residues 349through 351 of SEQ ID NO:54; and at least residues 369 through 371 ofSEQ ID NO:54. For example, the antibody specifically binds to an epitopethat contains at least residues 328, 329, 349 through 351 and 369through 371 of SEQ ID NO:54. In another example, the antibodyspecifically binds to an epitope that includes at least residues 293through 295, 296, 297 and 319 through 321 of SEQ ID NO:54.

Antibodies of the invention bind the TLR4/MD2 complex, wherein theantibody binds to an epitope on human MD-2 between residues 19 and 57 ofSEQ ID NO:44. For example, the antibody specifically binds to an epitopethat contains at least residues 53 of SEQ ID NO:44.

Antibodies of the invention also include humanized antibodies thatimmunospecifically bind a TLR4/MD-2 complex, wherein the antibodyexhibits greater than 50% inhibition of lipopolysaccharide (LPS)-inducedpro-inflammatory cytokine production in human TLR4/MD-2 transfectedHEK293 cells at a concentration of 1 μg/ml. For example, antibodies ofthe invention exhibit greater than 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 92%, 95%, 97%, 98%, or 99% inhibition of LPS-inducedpro-inflammatory cytokine production in human TLR4/MD-2 transfectedHEK293 cells at a concentration of 1 μg/ml. As used herein, the term“pro-inflammatory cytokine” refers to those immunoregulatory cytokinesthat promote inflammation and/or are associated with inflammation.Pro-inflammatory cytokines, include, for example, IL-6, IL-8, TNF-alpha,IL1-alpha, IL1-beta, IFN-alpha, IFN-beta, IFN-gamma, IL-10, IL12, IL-23,IL17, and IL18.

Antibodies of the invention, for example, inhibit LPS-inducedpro-inflammatory cytokine production at least two-fold, five-fold,10-fold, 20-fold, 50-fold, 75-fold, or 100-fold more than thecommercially available, anti-TLR4 non-blocking monoclonal antibodyHTA125.

The present invention also provides methods of treating or preventingpathologies associated with aberrant TLR4/MD-2 activation and/oraberrant LPS activity (e.g., aberrant pro-inflammatory cytokineproduction such as aberrant IL-8 production), or alleviating a symptomassociated with such pathologies, by administering a monoclonal antibodyof the invention (e.g., a murine monoclonal or humanized monoclonalantibody) to a subject in which such treatment or prevention is desired.The subject to be treated is, e.g., human. The monoclonal antibody isadministered in an amount sufficient to treat, prevent or alleviate asymptom associated with the pathology. The amount of monoclonal antibodysufficient to treat or prevent the pathology in the subject is, forexample, an amount that is sufficient to reduce LPS-induced productionof one or more pro-inflammatory cytokines (e.g., IL-8). As used herein,the term “reduced” refers to a decreased production of apro-inflammatory cytokine in the presence of a monoclonal antibody ofthe invention, wherein the production is, for example, localpro-inflammatory cytokine production (e.g., at a site of inflamedtissue) or systemic pro-inflammatory cytokine production. LPS-inducedproduction of a pro-inflammatory cytokine such as IL-8 is decreased whenthe level of pro-inflammatory cytokine (e.g., IL-8) production in thepresence of a monoclonal antibody of the invention is greater than orequal to 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%,99%, or 100% lower than a control level of pro-inflammatory cytokineproduction (i.e., the level of pro-inflammatory cytokine production inthe absence of the monoclonal antibody). Level of pro-inflammatorycytokine production (e.g., IL-8 or IL-6) is measured, e.g., using thehuman whole blood or huTLR4/MD2 transfected HEK293 cellular assaysdescribed herein. Those skilled in the art will appreciate that thelevel of pro-inflammatory cytokine production can be measured using avariety of assays, including, for example, commercially available ELISAkits.

Pathologies treated and/or prevented using the monoclonal antibodies ofthe invention (e.g., a murine monoclonal or humanized monoclonalantibody) include, for example, sepsis induced by microbial products,acute inflammation, chronic inflammation (e.g., chronic inflammationassociated with allergic conditions and asthma), autoimmune diseases(e.g., IBD and atherosclerosis) and diseases in which mechanical stressinduces the expression of endogenous soluble stress factors (e.g.,Hsp60, fibronectin, heparan sulphate, hyaluronan, gp96, β-Defensin-2 andsurfactant protein A). Pathologies in which mechanical stress inducesthe expression of endogenous soluble stress factors include, forexample, osteoarthritis and rheumatoid arthritis. Pathologies associatedwith mechanical stress can also occur in subjects and patients placed onrespirators, ventilators and other respiratory-assist devices. Suchpathologies include, for example, ventilator-induced lung injury(“VILI”), also referred to as ventilation-associated lung injury(“VALI”).

Pharmaceutical compositions according to the invention can include anantibody of the invention and a carrier. These pharmaceuticalcompositions can be included in kits, such as, for example, diagnostickits.

The present invention also provides soluble chimeric toll receptorproteins (also referred to herein as toll-like receptor proteins),methods for expressing toll receptor proteins, and methods for purifyingsuch proteins in a soluble form.

The present invention provides chimeric polypeptides in which atoll-like receptor polypeptide, or a biologically active derivativethereof, is operably linked to an MD accessory polypeptide, or abiologically active derivative thereof. The toll-like receptorpolypeptide is a polypeptide selected from the group consisting of TLRs1-10 and RP105.

The MD accessory polypeptide is, for example, MD-1 or MD-2. Thetoll-like receptor polypeptide is, in some instances, operably linked tothe MD accessory polypeptide using a flexible glycine-serine linker,which renders the toll receptor both stable during expression andsoluble during purification. For example, a chimeric polypeptide of theinvention includes the extracellular portion of a toll receptor fused atits C terminus to the N terminus of a mature MD protein (i.e., MD-1 orMD-2) via a flexible glycine/serine linker.

The present invention also provides methods for producing solublechimeric fusion proteins by coupling a toll-like receptor polypeptide,or a biologically active derivative thereof, to an MD accessorypolypeptide, or a biologically active derivative thereof. The presentinvention also provides methods for producing soluble chimeric fusionproteins by constructing a vector that includes a nucleic acid sequenceencoding a toll-like receptor polypeptide (or a biologically activederivative thereof) coupled to a nucleic acid sequence encoding an MDaccessory polypeptide (or a biologically active derivative thereof);transfecting a cell with this vector; culturing the cell underconditions that permit production of a fusion protein having a toll-likereceptor polypeptide coupled to an MD accessory polypeptide; andisolating that fusion protein. The MD accessory polypeptide is, forexample, MD-1 or MD-2, and the toll-like receptor polypeptide can be apolypeptide selected from the group consisting of TLRs 1-10 and RP105.The toll-like receptor polypeptide is operably linked to the MDaccessory polypeptide by a flexible glycine-serine linker, which rendersthe toll receptor both stable during expression and soluble duringpurification.

The present invention also provides methods of treating or preventingpathologies associated with aberrant toll-like receptor function, oralleviating a symptom associated with these pathologies, byadministering a soluble chimeric polypeptide of the invention to asubject in which such treatment or prevention or alleviation is desiredin an amount sufficient to treat or prevent or alleviate the pathology,or a symptom thereof, in the subject. The subject to be treated is,e.g., human. The amount of soluble chimeric polypeptide sufficient totreat or prevent the pathology in the subject is an amount that issufficient to modulate (e.g., reduce or prevent) the activation of atoll-like receptor in the subject to be treated. Activation of atoll-receptor is reduced or decreased when the level of toll-receptoractivation in the presence of a chimeric protein of the invention isgreater than or equal to 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%,75%, 80%, 90%, 95%, 99%, or 100% lower than a control level of toll-likereceptor activation (i.e., the level of activation the absence of thechimeric protein). The level of toll-receptor activation is measuredusing any of a variety of techniques known in the art. For example, thelevel of TLR4 activation can be measured by detecting the level ofLPS-induced IL-8 production. Those skilled in the art will appreciatethat the level of toll-receptor activation can also be measured, forexample, by detecting activation, if any, of NF-kappa B or JNK (c-junterminal kinase), which initiate the transcription of genes encodingpro-inflammatory cytokines (e.g., IL1-alpha, IL1-beta, IL6, andTNF-alpha). Activation of JNK and/or NF-kappa B can be detected bymeasuring the levels of one or more pro-inflammatory cytokines.

In some embodiments, the pathology to be treated is sepsis, acuteinflammation, chronic inflammation or an autoimmune disease. Forexample, the pathology is any one of a variety of types of arthritis.

The present invention also includes antibodies that immunospecificallybind to the soluble chimeric polypeptides of the invention, such as, forexample, monoclonal antibodies or humanized antibodies.

Pharmaceutical compositions according to the invention can include asoluble chimeric polypeptide of the invention and a carrier, and/or anantibody of the invention and a carrier. These pharmaceuticalcompositions can be included in kits, such as, for example, diagnostickits.

The invention also provides methods of screening for a ligand that bindsa toll-like receptor and modulates toll-like receptor activity.According to these methods of the invention, these ligands areidentified by providing a chimeric polypeptide of the invention that hasa property or function that is ascribable to that polypeptide;contacting the chimeric polypeptide with a candidate compound; anddetermining whether the candidate compound alters the property orfunction ascribable to the polypeptide, wherein an alteration in theproperty or function ascribable to the polypeptide in the presence ofthe candidate compound indicates that the candidate compound is a ligandthat modulates toll-like receptor activity.

One skilled in the art will appreciate that the chimeric polypeptidesand antibodies of the invention have a variety of uses. For example, thechimeric proteins of the invention are used as therapeutic agents toprevent the activation of TLRs in disorders such as, for example,sepsis, acute inflammation, chronic inflammation, autoimmune diseasesand various forms of arthritis. The chimeric proteins of the inventionare also used as immunogens in more efficient methods of generatingbinding and blocking anti-TLR antibodies, and/or these chimericpolypeptides can be used as reagents in assays that screen for smallmolecular weight binders and blockers of TLRs activity. The chimericproteins and/or antibodies of the invention are also used as reagents indiagnostic kits or as diagnostic tools, or these chimeric proteinsand/or antibodies can be used in competition assays to generatetherapeutic reagents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting the binding of a murine monoclonal antibody,referred to herein as “18H10”, to the TLR4/MD-2 complex. Specificity ofbinding is shown by flow cytometry using mock transfected or TLR4/MD-2transfected cells. The results using mock-transfected cells are shown inthe filled graph (left), while the results using TLR4/MD-2 transfectedcells are shown as in the outline graph (right).

FIG. 2 is a graph depicting inhibition of lipopolysaccharide(LPS)-induced IL-8 production in TLR4/MD-2 transfected HEK 293 cells bythe monoclonal antibody mu18H10. The cells were incubated with eithermu18H10, HTA 125 (a commercially available anti-human TLR4 non-blockingMAb) or an antibody control at the indicated concentrations andsubsequently incubated with LPS (15 ng/ml). IL-8 levels were assessed 16hours post LPS treatment.

FIG. 3 is a series of graphs depicting inhibition of LPS-induced IL-8production in human whole blood by the monoclonal antibody mu18H10.Whole blood was drawn from 3 healthy volunteers, treated with heparinand diluted 1:4 in RPMI medium. The following antibodies were added atthe concentrations indicated: control monoclonal antibody; HTA125 andmu18H10. LPS was subsequently added for a final concentration of 10ng/ml, and IL-8 levels were measured 6 hours post LPS treatment.

FIG. 4 is a series of graphs depicting the specificity of the mu18H 10monoclonal antibody for MD-2. The specificity of the mu18H10 antibody isshown by flow cytometry analysis of HEK 293 cells transientlytransfected with either human TLR4 and human MD-2 (Panels A, E and I);rabbit TLR4 and rabbit MD-2 (Panels B, F and J); human TLR4 and rabbitMD-2 (Panels C, G and K); or rabbit TLR4 and human MD-2 (Panels D, H andL). Cells were incubated with either α-FLAG™ antibody (to detect TLR4expression); α-C-myc antibody (to detect MD-2 expression) or the mu18H10monoclonal antibody, followed by an APC-coupled α-mouse (H+L) antibody.

FIG. 5A is a graph demonstrating the lack of specificity of mu18H10 forrecombinant soluble MD-2 purified from baculovirus-infected insect cellsupernatants as determined by ELISA. Protein was coated directly on96-well plates (5 μg/ml) followed by purified MAb at the indicatedconcentration and anti-mouse IgG (H+L) HRP.

FIG. 5B is a graph demonstrating that MD-2 must be associated with TLR4for the mu18H10 antibody to recognize it. Lysates (Panel 1, i.e., upperpanel) or supernatants (Panel 2, i.e., lower panel) from HEK 293 cells,transiently transfected as indicated, were incubated in wells coatedwith anti-FLAG M2. Binding of a biotinylated form of mu18H10 wasdetected using streptavidin-HRP. Biotinylated 12D4 (an anti-TLR4 MAb)with streptavidin-HRP or a polyclonal rabbit Ab raised against solubleMD-2 with an anti rabbit IgG-HRP controlled the presence of TLR4 andMD-2 respectively. In this experiment, TLR4 had a FLAG tag at theN-terminus and was expressed using the vector pCNDA3.1(−)hygro(Invitrogen). MD-2 had FLAG and 6×Histidine tags at the C terminus andwas expressed using the vector pCDNA3 (Invitrogen). Mock cells weretransfected with empty plasmid.

FIGS. 6A-6F are a series of illustrations depicting the VH nucleotidesequence (SEQ ID NO:1) (FIG. 6A), the VH amino acid sequence (SEQ IDNO:2) (FIG. 6B), the VL nucleotide sequence (SEQ ID NO:6) (FIG. 6D), andthe VL amino acid sequence (SEQ ID NO:7) for mu18H10 (FIG. 6E). The VHcomplementarity determining regions (CDRs) (SEQ ID NOs:3, 4 and 5) (FIG.6C) and the VL CDRs (SEQ ID NOs: 8, 9 and 10) (FIG. 6F) are highlightedin the underlined, italic text in FIGS. 6B and 6E.

FIG. 7 is a graph depicting that the VH and VL nucleotide sequence ofmu18H10 expressed as a chimeric MAb (“chimeric 18H10”) is capable ofbinding specifically to the human TLR4/MD-2 complex on the surface oftransfected CHO cells. MAb binding to the TLR4/MD-2 transfected CHOcells is shown by flow cytometry using chimeric 18H10 or an isotypematched control MAb at the concentrations indicated.

FIG. 8 is a graph depicting inhibition of lipopolysaccharide(LPS)-induced IL-8 production in TLR4/MD-2 transfected HEK 293 cells bythe chimeric 18H10 MAb. Cells were incubated with mu18H10, or chimeric18H10 at the indicated concentrations and subsequently incubated withLPS (15 ng/ml). IL-8 levels were assessed 16 hours post LPS-treatment.Inhibition of LPS-induced IL-8 production by the chimeric 18H10 wassimilar to the inhibition by the 18H10 mouse MAb of the invention.

FIG. 9 is a graph depicting the binding of a murine monoclonal antibody,referred to herein as “16G7”, to the TLR4/MD-2 complex. Specificity ofbinding is shown by flow cytometry using mock-transfected or TLR4/MD-2transfected cells. The results using mock transfected cells are shown inthe filled graph (left), while the results using TLR4/MD-2 transfectedcells are shown as in the outline graph (right).

FIG. 10 is a graph depicting inhibition of lipopolysaccharide(LPS)-induced IL-8 production in TLR4/MD-2 transfected HEK 293 cells bythe monoclonal antibody mu16G7. The cells were incubated with the mu16G7monoclonal antibody, the HTA 125 anti-TLR4 MAb or an antibody control atthe indicated concentrations and subsequently incubated with LPS (15ng/ml). IL-8 levels were assessed 16 hours post LPS treatment.

FIG. 11 is a series of graphs depicting inhibition of LPS-induced IL-8production in human whole blood by the monoclonal antibody mu16G7. Wholeblood was drawn from 3 healthy volunteers, treated with heparin anddiluted 1:4 in RPMI medium. The following antibodies were added at theconcentrations indicated: Isotype matched control; HTA125 (anti-humanTLR4 non-blocking monoclonal antibody); mu16G7 and 28C5 (anti-human CD14blocking monoclonal antibody). LPS was subsequently added for a finalconcentration of 10 ng/ml.

FIG. 12 is a series of graphs depicting the specificity of the mu16G7monoclonal antibody for TLR4. The specificity of the mu16G7 antibody isshown by flow cytometry analysis of HEK 293 cells transientlytransfected with either rabbit TLR4 and rabbit MD-2 (Panels A, E and I);human TLR4 and human MD-2 (Panels B, F and J); rabbit TLR4 and humanMD-2 (Panels C, G and K); or human TLR4 and rabbit MD-2 (Panels D, H andL). Cells were incubated with either α-FLAG™ antibody (to detect TLR4expression); α-C-myc antibody (to detect MD-2 expression) or the mu16G7monoclonal antibody, followed by an APC-coupled α-mouse (H+L) antibody.

FIGS. 13A-13F are a series of illustrations depicting the VH nucleotidesequence (SEQ ID NO:11) (FIG. 13A), the VH amino acid sequence (SEQ IDNO:12) (FIG. 13B), the VL nucleotide sequence (SEQ ID NO:16) (FIG. 13D),and the VL amino acid sequence (SEQ ID NO:17) (FIG. 13E) for mu16G7. TheVH complementarity determining regions (CDRs) (SEQ ID NOs: 13, 14 and15) (FIG. 13C) and the VL CDRs (SEQ ID NOs: 18, 19 and 20) (FIG. 13F)are highlighted in the underlined, italic text in FIGS. 13B and 13E.

FIG. 14 is a graph depicting the binding of a murine monoclonalantibody, referred to herein as “15C1”, to the TLR4/MD-2 complex.Specificity of binding is shown by flow cytometry using mock transfectedor TLR4/MD-2 transfected cells. The results using mock-transfected cellsare shown in the filled graph (left), while the results using TLR4/MD-2transfected cells are shown as in the outline graph (right).

FIG. 15 is a graph depicting inhibition of lipopolysaccharide(LPS)-induced IL-8 production in TLR4/MD-2 transfected HEK 293 cells bythe monoclonal antibody mu15C1. The cells were incubated with the mu15C1monoclonal antibody, HTA 125 (anti-human TLR4 non-blocking monoclonalantibody) and an isotype-matched control (IgG1) at the indicatedconcentrations and subsequently incubated with LPS (15 ng/ml). IL-8levels were assessed 16 hours post LPS treatment.

FIG. 16 is a series of graphs depicting inhibition of LPS-induced IL-6production in human whole blood by the monoclonal antibody mu15C1. Wholeblood was drawn from 3 healthy volunteers, treated with heparin anddiluted 1:4 in RPMI medium. The following antibodies were added at theconcentrations indicated: Isotype matched control (IgG1); HTA125(anti-human TLR4 non-blocking monoclonal antibody); mu15C1 and 28C5(anti-human CD14 blocking monoclonal antibody). LPS was subsequentlyadded for a final concentration of 10 ng/ml.

FIG. 17 is a series of graphs depicting the specificity of the mu15C1monoclonal antibody for TLR4. The specificity of the mu15C1 antibody isshown by flow cytometry analysis of HEK 293 cells transientlytransfected with either mock vector, i.e., empty vector (Panel A), humanTLR4 (Panel B), human TLR4 and human MD-2 (Panel C), rabbit TLR4 andrabbit MD-2 (Panel D), human TLR4 and rabbit MD-2 (Panel E), or rabbitTLR4 and human MD-2 (Panel F). Cells were incubated with the mu15C1monoclonal antibody (10 μg/ml), followed by an APC-coupled α-mouse (H+L)antibody.

FIGS. 18A-18F are a series of illustrations depicting the VH nucleotidesequence (SEQ ID NO:21) (FIG. 18A), the VH amino acid sequence (SEQ IDNO:22) (FIG. 18B), the VL nucleotide sequence (SEQ ID NO:26) (FIG. 18D),and the VL amino acid sequence (SEQ ID NO:27) (FIG. 18E) for mu15C1. TheVH complementarity determining regions (CDRs) (SEQ ID NOs: 23, 24 and25) (FIG. 18C) and the VL CDRs (SEQ ID NOs: 28, 29 and 30) (FIG. 18F)are highlighted in the underlined, italic text in FIGS. 18B and 18E.

FIG. 19 is a graph depicting that the VH and VL nucleotide sequence ofmu15C1 expressed as a chimeric MAb (“chimeric 15C1”) is capable ofbinding specifically to the human TLR4/MD-2 complex on the surface oftransfected CHO cells. MAb binding to the TLR4/MD-2 complex is shown byflow cytometry using chimeric 15C1 or an isotype matched controlmonoclonal antibody at the indicated concentration.

FIG. 20 is a graph depicting inhibition of lipopolysaccharide(LPS)-induced IL-8 production in TLR4/MD-2 transfected HEK 293 cells bythe chimeric 15C1 MAb. Cells were incubated with mu15C1 or chimerical15C1 at the concentrations indicated and subsequently incubated with LPS(15 ng/ml). IL-8 levels were assessed 16 hours post LPS treatment.Inhibition of LPS-induced IL-8 production by the chimeric 15C1 wassimilar to the inhibition by the mu15C1 mouse MAb of the invention.

FIG. 21 is a graph depicting the binding of a murine monoclonalantibody, referred to here in as “7E3”, to the TLR4/MD-2 complex.Specificity of binding is shown by flow cytometry using mock transfectedor TLR4/MD-2 transfected cells. The results using mock-transfected cellsare shown in the filled graph (left), while the results using TLR4/MD-2transfected cells are shown as in the outline graph (right).

FIG. 22 is a graph depicting inhibition of lipopolysaccharide(LPS)-induced IL-8 production in TLR4/MD-2 transfected HEK 293 cells bythe monoclonal antibody mu7E3. The cells were incubated with the mu7E3monoclonal antibody, HTA 125 (anti-human TLR4 non-blocking monoclonalantibody) and an isotype-matched control (IgG1) at the indicatedconcentrations and subsequently incubated with LPS (15 ng/ml). IL-8levels were assessed 16 hours post LPS treatment.

FIG. 23 is a series of graphs depicting inhibition of LPS-induced IL-6production in human whole blood by the monoclonal antibody mu7E3. Wholeblood was drawn from 3 healthy volunteers, treated with heparin anddiluted 1:4 in RPMI medium. The following antibodies were added at theconcentrations indicated: Isotype matched control (IgG1); HTA125(anti-human TLR4 non-blocking monoclonal antibody); mu7E3 and 28C5(anti-human CD14 blocking monoclonal antibody). LPS was subsequentlyadded for a final concentration of 10 ng/ml.

FIG. 24 is a series of graphs depicting the specificity of the mu7E3monoclonal antibody for the TLR4/MD-2 complex. The specificity of themu7E3 antibody is shown by flow cytometry analysis of HEK 293 cellstransiently transfected with either mock vector (Panel A), human TLR4(Panel B), human TLR4 and human MD-2 (Panel C), rabbit TLR4 and rabbitMD-2 (Panel D), human TLR4 and rabbit MD-2 (Panel E),or rabbit TLR4 andhuman MD-2 (Panel F). Cells were incubated with the mu7E3 monoclonalantibody (10 μg/ml), followed by an APC-coupled α-mouse (H+L) antibody.

FIGS. 25A-25F are a series of illustrations depicting the VH nucleotidesequence (SEQ ID NO:31) (FIG. 25A), the VH amino acid sequence (SEQ IDNO:32) (FIG. 25B), the VL nucleotide sequence (SEQ ID NO:36) (FIG. 25D),and the VL amino acid sequence (SEQ ID NO:37) (FIG. 25E) for mu7E3. TheVH complementarity determining regions (CDRs) (SEQ ID NOs: 33, 34 and35) (FIG. 25C) and the VL CDRs (SEQ ID NOs: 38, 39 and 40) (FIG. 25F)are highlighted in the underlined italic text in FIGS. 25B and 25E.

FIG. 26 is a graph illustrating that the VH and VL nucleotide sequenceof mu7E3 expressed as a chimeric MAb (“chimeric 7E3”) is capable ofbinding specifically to the human TLR4/MD-2 complex on the surface oftransfected CHO cells. Monoclonal antibody binding to TLR4/MD-2transfected CHO cells is shown by flow cytometry using chimeric 7E3 oran isotype matched control MAb at the indicated concentrations.

FIG. 27 is a graph depicting inhibition of lipopolysaccharide(LPS)-induced IL-8 production in TLR4/MD-2 transfected HEK 293 cells bythe chimeric 7E3 MAb. Cells were incubated with chimeric 7E3 or anisotype matched MAb control at the indicated concentrations andsubsequently incubated with LPS (15 ng/ml). IL-8 levels were assessed 16hours post LPS-treatment.

FIG. 28 is an illustration depicting the construction of a TLR4/MD-2fusion protein cDNA according to the present invention.

FIG. 29 is an illustration depicting the expression of a TLR4/MD-2chimeric protein of the invention in Sf9 cell lysates and supernatant.

FIG. 30 is an illustration depicting the purification of a TLR4/MD-2chimeric protein according to the invention from infected Sf9 celllysates.

FIG. 31 is a graph depicting the inhibition of lipopolysaccharide-(LPS)induced IL-8 production using a soluble chimeric TLR4/MD-2 proteinaccording to the present invention.

FIG. 32A illustrates a nucleic acid sequence encoding the accessoryprotein MD-1 (SEQ ID NO:41).

FIG. 32B depicts an amino acid sequence of a mature MD-1 accessoryprotein in a preferred embodiment of the invention (SEQ ID NO:42).

FIG. 33A illustrates a nucleic acid sequence encoding the accessoryprotein MD-2 (SEQ ID NO:43).

FIG. 33B depicts an amino acid sequence of a mature MD-2 accessoryprotein (SEQ ID NO:44).

FIGS. 34A, 34B and 34C are a schematic representation and a series ofgraphs depicting the binding of hu15C1 and hu7E3 to human-mouse hybridversions of TLR4. FIG. 34A is a schematic representation and summarytable of the mouse-human TLR4 hybrid mutants and antibody binding tothese mutants. Red regions in the schematic representation depictmouse-derived sequence and the blue regions represent human-derivedsequence. In the summary table of antibody binding, (++) representsstrong binding, (+) represents intermediate binding and (−) indicatesweak or no binding. FIG. 34B is a series of flow cytometry histogramsdepicting monoclonal antibody binding to transfected cells expressingthe human-mouse hybrids. The HEK 293 cells were transfected withwild-type TLR4 (row 1); mouse-human-human-human (MHHH) TLR4 (row 2);mouse-mouse-human-human (row 3); mouse-human-mouse-human (MHMH) TLR4(row 4) or human-human-human-mouse (HHHM) TLR4 (row 5). Cells wereincubated with either α-FLAG™ antibody (to detect TLR4 expression);α-C-myc antibody (to detect MD-2 expression) or the hu15C1 or hu7E3monoclonal antibody, followed by an APC-conjugated antibody. FIG. 34C isa series of flow cytometry histograms depicting monoclonal antibodybinding to transfected cells expressing the human-mouse hybrids.

FIGS. 35A and 35B are a schematic representation and a series of graphsdepicting binding of hu15C1 and hu7E3 to “fine-resolution” human-mousehybrid versions of TLR4. FIG. 35A is a schematic representation andsummary table of the “fine resolution” mouse-human TLR4 hybrid mutantsand antibody binding to these mutants. Red regions representmouse-derived sequence and blue regions represent human-derivedsequence. In the summary table of antibody binding, (++) representsstrong binding, (+) represents intermediate binding and (−) indicatesweak or no binding. FIG. 35B is a series of flow cytometry histogramsdepicting MAb binding to transfected cells expressing the human-mousehybrids.

FIGS. 36A and 36B are a schematic representation and a series of graphsdepicting binding of hu15C1 and hu7E3 to alanine-scanning mutants ofTLR4. FIG. 36A is a schematic representation of the alanine scanningmutants (QC1-QC20; boxed from 1 to 20 on the human sequence) selectedafter alignment of the human and mouse TLR4 amino acid sequences fromamino acids 289-375 and amino acids 288-373, respectively. Mutants weredesigned so that any amino acid differences between human and mousesequences within the boxes were converted to an alanine in the humansequence (e.g., QC2 is modified from YL to AA). FIG. 36B is a series ofbar graphs representing MAb binding to transfected cells expressing theTLR4 alanine-scan mutants. For C-myc, the actual MFI obtained followingflow cytometric analysis is shown. For hu18H10, hu15C1 and hu7E3, valuesrepresent “normalized” antibody binding by dividing the MFI obtained forthe given MAb by that obtained for the C-myc.

FIGS. 37A and 37B are a schematic representation and a series of graphsdepicting binding of hu18H10 to human-mouse hybrid versions of MD-2.FIG. 37A is a schematic representation and summary table of themouse-human MD-2 hybrid mutants and antibody binding to these mutants.Red regions represent mouse-derived sequence and blue regions representhuman-derived sequence. In the summary table of antibody binding, (++)represents strong binding, (+) represents intermediate binding and (−)indicates weak or no binding. FIG. 37B is a series of flow cytometryhistograms depicting MAb binding to transfected cells expressing thehuman-mouse hybrids.

FIGS. 38A and 38B are a schematic representation and a series of graphsdepicting binding of hu18H10 to alanine-scanning mutants of MD-2. FIG.38A is a schematic representation of the alanine scanning mutants(QC1-QC14; boxed from 1 to 14 on the human sequence) selected afteralignment of the human and mouse MD-2 amino acid sequences from aminoacids 19-57. Mutants were designed so that any amino acid differencesbetween human and mouse sequences were converted to an alanine in thehuman sequence (e.g., QC1 is modified from Q to A). FIG. 38B is a seriesof bar graphs representing MAb binding to transfected cellsco-expressing wt TLR4 along with the MD-2 mutants. For the anti-6×HISand hu15C1 MAbs, the actual MFI obtained following flow cytometricanalysis is shown. For hu18H10, both actual MFIs and values represent“normalized” antibody binding (by dividing the MFI obtained for the MAbby that obtained for anti-6×HIS) are shown.

FIG. 39 is a graph depicting that the hu18H10 humanized monoclonalantibody (“18H10 hum”) is capable of binding specifically to the humanTLR4/MD-2 complex on the surface of transfected CHO cells. MAb bindingto the TLR4/MD-2 transfected CHO cells is shown by flow cytometry usingthe hu18H10 antibody or the chimeric 18H10 (“18H10chim”) (describedabove) at the concentrations indicated. Binding is measured as acellular Mean Fluorescence Intensity (MFI) value.

FIG. 40 is a graph depicting that the hu7E3 humanized monoclonalantibody that includes V_(H) 2-70 shown in SEQ ID NO:51 (“7E32-70/L-19”) and the hu7E3 humanized monoclonal antibody that includesV_(H) 3-66 (SEQ ID NO:52) (“7E3 3-66/L19”) are capable of bindingspecifically to the human TLR4/MD-2 complex on the surface oftransfected CHO cells. MAb binding to the TLR4/MD-2 transfected CHOcells is shown by flow cytometry using the hu7E3 antibodies or thechimeric 7E3 (“7E3 CHIM”) (described above) at the concentrationsindicated. Binding is measured as a cellular Mean Fluorescence Intensity(MFI) value.

FIG. 41 is a graph depicting that the hu1 C1 humanized antibody thatincludes V_(H) 4-28 shown in SEQ ID NO:45 (“15C14-28/A26”) and variantsthereof in which residues at chosen positions have been replaced by thecorresponding amino acid in the given human germline(“15C1 4-28 QC30/A26”; “15C1 4-28 QC 48/A26”; “15C1 4-28 QC 67/A26” and “15C1 4-28 QC69/A26”, see TABLE 1) are capable of binding specifically to the humanTLR4/MD-2 complex on the surface of transfected CHO cells. MAb bindingto the TLR4/MD-2 transfected CHO cells is shown by flow cytometry usingthe hu15C1 antibodies or the chimeric 15C1 (“15C1 CHIM”) (describedabove) at the concentrations indicated. Binding is measured as acellular Mean Fluorescence Intensity (MFI) value.

FIG. 42 is a graph depicting that the hu15C1 humanized antibody thatincludes V_(H) 3-66 shown in SEQ ID NO:46 and V_(L) L6 shown in SEQ IDNO:47 (15C1 3-66 L6) and the hu15C1 humanized antibody that includesV_(H) 3-66 shown in SEQ ID NO:46 and V_(L) A26 shown in SEQ ID NO:48(15C1 3-66 A26) are capable of binding specifically to the humanTLR4/MD-2 complex on the surface of transfected CHO cells. MAb bindingto the TLR4/MD-2 transfected CHO cells is shown by flow cytometry usingthe hu15C1 3-66 L6 and hu15C1 3-66 A26 hu15C1 antibodies, the hu15C14-28 A26 antibody or the chimeric 15C1 (“15C1 CHIM”) (described above)at the concentrations indicated. Binding is measured as a cellular MeanFluorescence Intensity (MFI) value.

FIG. 43 depicts the amino acid sequence of human toll-like receptor 4(TLR4).

FIG. 44 is a series of graphs depicting inhibition of LPS-induced IL-6production in human whole blood by the monoclonal antibody hu15C1 havingthe heavy chain variable region 4-28 and the light chain variable regionA26 (“4-28/A26”). The hu15C1 4-28/A26 was compared to an isotype matchedcontrol (IgG1) and the 15C1 chimeric antibody described herein.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides monoclonal antibodies (MAbs) thatspecifically bind the human TLR4/MD-2 receptor complex. This receptorcomplex is activated by lipopolysaccharide (LPS), the major component ofthe outer membrane of gram-negative bacteria. The monoclonal antibodiesof the invention inhibit receptor activation and subsequentintracellular signaling via LPS. Thus, the monoclonal antibodiesneutralize the activation of the TLR4/MD-2 receptor complex. Inparticular, the invention provides monoclonal antibodies that recognizethe TLR4/MD-2 receptor complex expressed on the cell surface. Thesemonoclonal antibodies block LPS-induced IL-8 production. In addition,some monoclonal antibodies of the invention also recognize TLR4 when notcomplexed with MD-2. The monoclonal antibody is, e.g., a humanizedantibody.

Antibodies of the invention include antibodies that bind the humanTLR4/MD-2 receptor complex and also bind TLR4 independently of thepresence of MD-2. Antibodies of the invention also include antibodiesthat bind the TLR4 portion of the human TLR4/MD-2 receptor complex butbinding is dependent on the presence of MD-2, but binding is greatlyenhanced by the presence of MD-2, which suggests that the presence ofthe MD-2 causes a conformational change in TLR4, thereby exposing anepitope bound by the antibody. In addition, antibodies of the inventioninclude antibodies that bind the human TLR4/MD-2 receptor complex andalso bind MD-2 in the presence of TLR4.

Antibodies of the invention immunospecifically bind a TLR4/MD-2 complex,wherein the antibody binds to an epitope that includes one or more aminoacid residues on human TLR4 between residues 289 and 375 of SEQ IDNO:54. Antibodies of the invention immunospecifically bind the TLR4/MD2complex, wherein the antibody binds to an epitope on human MD-2 betweenresidues 19 and 57 of SEQ ID NO:44.

Exemplary antibodies of the invention include, for example, the 18H10antibody, the 16G7 antibody, the 15C1 antibody and the 7E3 antibody.These antibodies show specificity for the human TLR4/MD-2 receptorcomplex, and they have been shown to inhibit receptor activation andsubsequent intracellular signaling via LPS. These antibodies havedistinct specificities. For example, 16G7 and 15C1 bind TLR4independently of the presence of MD-2, 7E3 binds to TLR4, but binding isdependent on the presence of MD-2, and 18H10 binds to MD-2, but requiresthe presence of TLR4, as the MAb does not bind soluble forms of MD-2.

As used herein, the terms “16G7”, “mu16G7”, “7E3”, “mu7E3”, “15C1”,“mu15C1”, “18H10” or “mu18H10” refer to the murine monoclonal antibody,and the terms “hu7E3”, “hu15C1”, or “hu18H10” refer to the humanizedmonoclonal antibody.

The present invention also provides soluble chimeric toll receptorproteins (also referred to herein as toll-like receptor proteins),methods for expressing toll receptor proteins, and methods for purifyingsuch proteins in a soluble form. The chimeric proteins are useful, e.g.,in generating antibodies.

TLRs recognize microbial particles and activate immune cells against thesource of these microbial particles. (See Takeda et al., Annu. Rev.Immunol., 21: 335-76 (2003), hereby incorporated by reference in itsentirety). TLR4 and MD-2 have been shown to form a complex on the cellsurface, and the presence of MD-2 appears essential for theresponsiveness of TLR4 to various ligands, including LPS. LPS is agram-negative bacterial outer membrane glycolipid that is capable ofstrongly activating the innate immune system. LPS has been implicated asone of the major factors activating the immune system during the severegeneralized inflammation resulting from gram-negative infection.(Lakhani et al., Curr. Opin. Pediatr. 15: 278-282 (2003), herebyincorporated by reference in its entirety).

LPS delivers signals to immune cells via its multi-chain receptor inwhich the TLR4/MD-2 complex is the principle signaling component. LPShas been shown to exert its effects on the immune system via signalingthrough TLR4. LPS rapidly binds to the lipopolysaccharide-bindingprotein (LBP) in the bloodstream, and in this form, LPS interacts withthe GPI-anchored cell surface protein CD14. LPS is then transferred toTLR4, which transduces an intracellular activation signal. Anotherprotein, MD-2, has been found to be necessary for signal transductionvia TLR4 to occur. MD-2 interacts directly with TLR4 and plays animportant role in its post-translational modification and intracellulartrafficking. In addition, MD-2 has been shown to directly bind LPS,which demonstrates the importance of this accessory protein in the LPSreceptor complex. (See Miyake K., Int. Immunopharmacol. 3:119-128(2003), hereby incorporated by reference in its entirety).

Accordingly, neutralization of LPS signaling mediated by the TLR4/MD-2complex is a potential therapeutic strategy in the treatment ofdisorders such as, for example, acute systemic inflammation and sepsisinduced by gram-negative bacterial infection.

Definitions:

Unless otherwise defined, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures utilized in connection with, and techniques of, cell andtissue culture, molecular biology, and protein and oligo- orpolynucleotide chemistry and hybridization described herein are thosewell known and commonly used in the art. Standard techniques are usedfor recombinant DNA, oligonucleotide synthesis, and tissue culture andtransformation (e.g., electroporation, lipofection). Enzymatic reactionsand purification techniques are performed according to manufacturer'sspecifications or as commonly accomplished in the art or as describedherein. The foregoing techniques and procedures are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification See e.g., Sambrook et al.Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclaturesutilized in connection with, and the laboratory procedures andtechniques of, analytical chemistry, synthetic organic chemistry, andmedicinal and pharmaceutical chemistry described herein are those wellknown and commonly used in the art. Standard techniques are used forchemical syntheses, chemical analyses, pharmaceutical preparation,formulation, and delivery, and treatment of patients.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

As used herein, the term “antibody” refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin (Ig) molecules,i.e., molecules that contain an antigen binding site that specificallybinds (immunoreacts with) an antigen. By “specifically bind” or“immunoreacts with” or “immunospecifically bind” is meant that theantibody reacts with one or more antigenic determinants of the desiredantigen and does not react with other polypeptides or binds at muchlower affinity (K_(d)>10⁻⁶). Antibodies include, but are not limited to,polyclonal, monoclonal, chimeric, dAb (domain antibody), single chain,F_(ab), F_(ab′) and F_((ab′)2) fragments, scFvs, and an F_(ab)expression library.

The basic antibody structural unit is known to comprise a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function. Ingeneral, antibody molecules obtained from humans relate to any of theclasses IgG, IgM, IgA, IgE and IgD, which differ from one another by thenature of the heavy chain present in the molecule. Certain classes havesubclasses as well, such as IgG₁, IgG₂, and others. Furthermore, inhumans, the light chain may be a kappa chain or a lambda chain.

The term “monoclonal antibody” (MAb) or “monoclonal antibodycomposition”, as used herein, refers to a population of antibodymolecules that contain only one molecular species of antibody moleculeconsisting of a unique light chain gene product and a unique heavy chaingene product. In particular, the complementarity determining regions(CDRs) of the monoclonal antibody are identical in all the molecules ofthe population. MAbs contain an antigen binding site capable ofimmunoreacting with a particular epitope of the antigen characterized bya unique binding affinity for it.

The term “antigen-binding site,” or “binding portion” refers to the partof the immunoglobulin molecule that participates in antigen binding. Theantigen binding site is formed by amino acid residues of the N-terminalvariable (“V”) regions of the heavy (“H”) and light (“L”) chains. Threehighly divergent stretches within the V regions of the heavy and lightchains, referred to as “hypervariable regions,” are interposed betweenmore conserved flanking stretches known as “framework regions,” or“FRs”. Thus, the term “FR” refers to amino acid sequences which arenaturally found between, and adjacent to, hypervariable regions inimmunoglobulins. In an antibody molecule, the three hypervariableregions of a light chain and the three hypervariable regions of a heavychain are disposed relative to each other in three dimensional space toform an antigen-binding surface. The antigen-binding surface iscomplementary to the three-dimensional surface of a bound antigen, andthe three hypervariable regions of each of the heavy and light chainsare referred to as “complementarity-determining regions,” or “CDRs.” Theassignment of amino acids to each domain is in accordance with thedefinitions of Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health, Bethesda, Md. (1987 and 1991)), orChothia & Lesk J. Mol. Biol. 196:901-917 (1987), Chothia et al. Nature342:878-883 (1989).

As used herein, the term “epitope” includes any protein determinantcapable of specific binding to an immunoglobulin, an scFv, or a T-cellreceptor. The term “epitope” includes any protein determinant capable ofspecific binding to an immunoglobulin or T-cell receptor. Epitopicdeterminants usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains and usually havespecific three dimensional structural characteristics, as well asspecific charge characteristics; For example, antibodies may be raisedagainst N-terminal or C-terminal peptides of a polypeptide. An antibodyis said to specifically bind an antigen when the dissociation constantis ≦1 μM; preferably ≦100 nM and most preferably ≦10 nM.

As used herein, the terms “immunological binding,” and “immunologicalbinding properties” refer to the non-covalent interactions of the typewhich occur between an immunoglobulin molecule and an antigen for whichthe immunoglobulin is specific. The strength, or affinity ofimmunological binding interactions can be expressed in terms of thedissociation constant (K_(d)) of the interaction, wherein a smallerK_(d) represents a greater affinity. Immunological binding properties ofselected polypeptides can be quantified using methods well known in theart. One such method entails measuring the rates of antigen-bindingsite/antigen complex formation and dissociation, wherein those ratesdepend on the concentrations of the complex partners, the affinity ofthe interaction, and geometric parameters that equally influence therate in both directions. Thus, both the “on rate constant” (K_(on)) andthe “off rate constant” (K_(off)) can be determined by calculation ofthe concentrations and the actual rates of association and dissociation.(See Nature 361:186-87 (1993)). The ratio of K_(off)/K_(on) enables thecancellation of all parameters not related to affinity, and is equal tothe dissociation constant K_(d). (See, generally, Davies et al. (1990)Annual Rev Biochem 59:439-473). An antibody of the present invention issaid to specifically bind to the Toll-like Receptor 4 (TLR4)/MD-2complex or to TLR4 when not complexed to MD-2, when the equilibriumbinding constant (K_(d)) is ≦1 μM, preferably ≦100 nM, more preferably≦10 nM, and most preferably ≦100 pM to about 1 pM, as measured by assayssuch as radioligand binding assays or similar assays known to thoseskilled in the art.

The term “isolated polynucleotide” as used herein shall mean apolynucleotide of genomic, cDNA, or synthetic origin or some combinationthereof, which by virtue of its origin the “isolated polynucleotide” (1)is not associated with all or a portion of a polynucleotide in which the“isolated polynucleotide” is found in nature, (2) is operably linked toa polynucleotide which it is not linked to in nature, or (3) does notoccur in nature as part of a larger sequence. Polynucleotides inaccordance with the invention include the nucleic acid moleculesencoding the heavy chain immunoglobulin molecules presented in SEQ IDNOS: 2, 12, 22, 32, 45, 46, 49, 51 and 52, and nucleic acid moleculesencoding the light chain immunoglobulin molecules represented in SEQ IDNOS: 7, 17, 27, 37, 47, 48, 50 and 53.

The term “isolated protein” referred to herein means a protein of cDNA,recombinant RNA, or synthetic origin or some combination thereof, whichby virtue of its origin, or source of derivation, the “isolated protein”(1) is not associated with proteins found in nature, (2) is free ofother proteins from the same source, e.g., free of marine proteins, (3)is expressed by a cell from a different species, or (4) does not occurin nature.

The term “polypeptide” is used herein as a generic term to refer tonative protein, fragments, or analogs of a polypeptide sequence. Hence,native protein fragments, and analogs are species of the polypeptidegenus. Polypeptides in accordance with the invention comprise the heavychain immunoglobulin molecules represented in SEQ ID NOS: 2, 12, 22, 32,45, 46, 49, 51 and 52, and the light chain immunoglobulin moleculesrepresented in SEQ ID NOS: 7, 17, 27, 37, 47, 48, 50 and 53, as well asantibody molecules formed by combinations comprising the heavy chainimmunoglobulin molecules with light chain immunoglobulin molecules, suchas kappa light chain immunoglobulin molecules, and vice versa, as wellas fragments and analogs thereof.

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory orotherwise is naturally-occurring.

The term “operably linked” as used herein refers to positions ofcomponents so described are in a relationship permitting them tofunction in their intended manner. A control sequence “operably linked”to a coding sequence is ligated in such a way that expression of thecoding sequence is achieved under conditions compatible with the controlsequences.

The term “control sequence” as used herein refers to polynucleotidesequences which are necessary to effect the expression and processing ofcoding sequences to which they are ligated. The nature of such controlsequences differs depending upon the host organism in prokaryotes, suchcontrol sequences generally include promoter, ribosomal binding site,and transcription termination sequence in eukaryotes, generally, suchcontrol sequences include promoters and transcription terminationsequence. The term “control sequences” is intended to include, at aminimum, all components whose presence is essential for expression andprocessing, and can also include additional components whose presence isadvantageous, for example, leader sequences and fusion partnersequences. The term “polynucleotide” as referred to herein means apolymeric boron of nucleotides of at least 10 bases in length, eitherribonucleotides or deoxynucleotides or a modified form of either type ofnucleotide. The term includes single and double stranded forms of DNA.

The term oligonucleotide referred to herein includes naturallyoccurring, and modified nucleotides linked together by naturallyoccurring, and non-naturally occurring oligonucleotide linkages.Oligonucleotides are a polynucleotide subset generally comprising alength of 200 bases or fewer. Preferably oligonucleotides are 10 to 60bases in length and most preferably 12, 13, 14, 15, 16, 17, 18, 19, or20 to 40 bases in length. Oligonucleotides are usually single stranded,e.g., for probes, although oligonucleotides may be double stranded,e.g., for use in the construction of a gene mutant. Oligonucleotides ofthe invention are either sense or antisense oligonucleotides.

The term “naturally occurring nucleotides” referred to herein includesdeoxyribonucleotides and ribonucleotides. The term “modifiednucleotides” referred to herein includes nucleotides with modified orsubstituted sugar groups and the like. The term “oligonucleotidelinkages” referred to herein includes Oligonucleotides linkages such asphosphorothioate, phosphorodithioate, phosphoroselerloate,phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate,phosphoronmidate, and the like. See e.g., LaPlanche et al. Nucl. AcidsRes. 14:9081 (1986); Stec et al. J. Am. Chem. Soc. 106:6077 (1984),Stein et al. Nucl. Acids Res. 16:3209 (1988), Zon et al. Anti CancerDrug Design 6:539 (1991); Zon et al. Oligonucleotides and Analogues: APractical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford UniversityPress, Oxford England (1991)); Stec et al. U.S. Pat. No. 5,151,510;Uhlmann and Peyman Chemical Reviews 90:543 (1990). An oligonucleotidecan include a label for detection, if desired.

The term “selectively hybridize” referred to herein means to detectablyand specifically bind. Polynucleotides, oligonucleotides and fragmentsthereof in accordance with the invention selectively hybridize tonucleic acid strands under hybridization and wash conditions thatminimize appreciable amounts of detectable binding to nonspecificnucleic acids. High stringency conditions can be used to achieveselective hybridization conditions as known in the art and discussedherein. Generally, the nucleic acid sequence homology between thepolynucleotides, oligonucleotides, and fragments of the invention and anucleic acid sequence of interest will be at least 80%, and moretypically with preferably increasing homologies of at least 85%, 90%,95%, 99%, and 100%. Two amino acid sequences are homologous if there isa partial or complete identity between their sequences. For example, 85%homology means that 85% of the amino acids are identical when the twosequences are aligned for maximum matching. Gaps (in either of the twosequences being matched) are allowed in maximizing matching gap lengthsof 5 or less are preferred with 2 or less being more preferred.Alternatively and preferably, two protein sequences (or polypeptidesequences derived from them of at least 30 amino acids in length) arehomologous, as this term is used herein, if they have an alignment scoreof at more than 5 (in standard deviation units) using the program ALIGNwith the mutation data matrix and a gap penalty of 6 or greater. SeeDayhoff, M. O., in Atlas of Protein Sequence and Structure, pp. 101-110(Volume 5, National Biomedical Research Foundation (1972)) andSupplement 2 to this volume, pp. 1-10. The two sequences or partsthereof are more preferably homologous if their amino acids are greaterthan or equal to 50% identical when optimally aligned using the ALIGNprogram. The term “corresponds to” is used herein to mean that apolynucleotide sequence is homologous (i.e., is identical, not strictlyevolutionarily related) to all or a portion of a referencepolynucleotide sequence, or that a polypeptide sequence is identical toa reference polypeptide sequence. In contradistinction, the term“complementary to” is used herein to mean that the complementarysequence is homologous to all or a portion of a reference polynucleotidesequence. For illustration, the nucleotide sequence “TATAC” correspondsto a reference sequence “TATAC” and is complementary to a referencesequence “GTATA”.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotide or amino acid sequences: “referencesequence”, “comparison window”, “sequence identity”, “percentage ofsequence identity”, and “substantial identity”. A “reference sequence”is a defined sequence used as a basis for a sequence comparison areference sequence may be a subset of a larger sequence, for example, asa segment of a full-length cDNA or gene sequence given in a sequencelisting or may comprise a complete cDNA or gene sequence. Generally, areference sequence is at least 18 nucleotides or 6 amino acids inlength, frequently at least 24 nucleotides or 8 amino acids in length,and often at least 48 nucleotides or 16 amino acids in length. Since twopolynucleotides or amino acid sequences may each (1) comprise a sequence(i.e., a portion of the complete polynucleotide or amino acid sequence)that is similar between the two molecules, and (2) may further comprisea sequence that is divergent between the two polynucleotides or aminoacid sequences, sequence comparisons between two (or more) molecules aretypically performed by comparing sequences of the two molecules over a“comparison window” to identify and compare local regions of sequencesimilarity. A “comparison window”, as used herein, refers to aconceptual segment of at least 18 contiguous nucleotide positions or 6amino acids wherein a polynucleotide sequence or amino acid sequence maybe compared to a reference sequence of at least 18 contiguousnucleotides or 6 amino acid sequences and wherein the portion of thepolynucleotide sequence in the comparison window may comprise additions,deletions, substitutions, and the like (i.e., gaps) of 20 percent orless as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted by the local homology algorithm of Smith and Waterman Adv.Appl. Math. 2:482 (1981), by the homology alignment algorithm ofNeedleman and Wunsch J. Mol. Biol. 48:443 (1970), by the search forsimilarity method of Pearson and Lipman Proc. Natl. Acad. Sci. (U.S.A.)85:2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage Release 7.0, (Genetics Computer Group, 575 Science Dr., Madison,Wis.), Geneworks, or MacVector software packages), or by inspection, andthe best alignment (i.e., resulting in the highest percentage ofhomology over the comparison window) generated by the various methods isselected.

The term “sequence identity” means that two polynucleotide or amino acidsequences are identical (i.e., on a nucleotide-by-nucleotide orresidue-by-residue basis) over the comparison window. The term“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U or I) or residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the comparison window (i.e., the windowsize), and multiplying the result by 100 to yield the percentage ofsequence identity. The terms “substantial identity” as used hereindenotes a characteristic of a polynucleotide or amino acid sequence,wherein the polynucleotide or amino acid comprises a sequence that hasat least 85 percent sequence identity, preferably at least 90 to 95percent sequence identity, more usually at least 99 percent sequenceidentity as compared to a reference sequence over a comparison window ofat least 18 nucleotide (6 amino acid) positions, frequently over awindow of at least 24-48 nucleotide (8-16 amino acid) positions, whereinthe percentage of sequence identity is calculated by comparing thereference sequence to the sequence which may include deletions oradditions which total 20 percent or less of the reference sequence overthe comparison window. The reference sequence may be a subset of alarger sequence.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis (2ndEdition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland7 Mass. (1991)). Stereoisomers (e.g., D-amino acids) of thetwenty conventional amino acids, unnatural amino acids such as α-,α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and otherunconventional amino acids may also be suitable components forpolypeptides of the present invention. Examples of unconventional aminoacids include: 4 hydroxyproline, γ-carboxyglutamate,ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine,N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,σ-N-methylarginine, and other similar amino acids and imino acids (e.g.,4-hydroxyproline). In the polypeptide notation used herein, theleft-hand direction is the amino terminal direction and the right-handdirection is the carboxy-terminal direction, in accordance with standardusage and convention.

Similarly, unless specified otherwise, the left-hand end ofsingle-stranded polynucleotide sequences is the 5′ end the left-handdirection of double-stranded polynucleotide sequences is referred to asthe 5′ direction. The direction of 5′ to 3′ addition of nascent RNAtranscripts is referred to as the transcription direction sequenceregions on the DNA strand having the same sequence as the RNA and whichare 5′ to the 5′ end of the RNA transcript are referred to as “upstreamsequences”, sequence regions on the DNA strand having the same sequenceas the RNA and which are 3′ to the 3′ end of the RNA transcript arereferred to as “downstream sequences”.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity, preferably at least 90 percent sequence identity,more preferably at least 95 percent sequence identity, and mostpreferably at least 99 percent sequence identity.

Preferably, residue positions which are not identical differ byconservative amino acid substitutions.

Conservative amino acid substitutions refer to the interchangeability ofresidues having similar side chains. For example, a group of amino acidshaving aliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine valine,glutamic-aspartic, and asparagine-glutamine.

As discussed herein, minor variations in the amino acid sequences ofantibodies or immunoglobulin molecules are contemplated as beingencompassed by the present invention, providing that the variations inthe amino acid sequence maintain at least 75%, more preferably at least80%, 90%, 95%, and most preferably 99%. In particular, conservativeamino acid replacements are contemplated. Conservative replacements arethose that take place within a family of amino acids that are related intheir side chains. Genetically encoded amino acids are generally dividedinto families: (1) acidic amino acids are aspartate, glutamate; (2)basic amino acids are lysine, arginine, histidine; (3) non-polar aminoacids are alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan, and (4) uncharged polar amino acids are glycine,asparagine, glutamine, cysteine, serine, threonine, tyrosine. Thehydrophilic amino acids include arginine, asparagine, aspartate,glutamine, glutamate, histidine, lysine, serine, and threonine. Thehydrophobic amino acids include alanine, cysteine, isoleucine, leucine,methionine, phenylalanine, proline, tryptophan, tyrosine and valine.Other families of amino acids include (i) serine and threonine, whichare the aliphatic-hydroxy family; (ii) asparagine and glutamine, whichare the amide containing family; (iii) alanine, valine, leucine andisoleucine, which are the aliphatic family; and (iv) phenylalanine,tryptophan, and tyrosine, which are the aromatic family. For example, itis reasonable to expect that an isolated replacement of a leucine withan isoleucine or valine, an aspartate with a glutamate, a threonine witha serine, or a similar replacement of an amino acid with a structurallyrelated amino acid will not have a major effect on the binding orproperties of the resulting molecule, especially if the replacement doesnot involve an amino acid within a framework site. Whether an amino acidchange results in a functional peptide can readily be determined byassaying the specific activity of the polypeptide derivative. Assays aredescribed in detail herein. Fragments or analogs of antibodies orimmunoglobulin molecules can be readily prepared by those of ordinaryskill in the art. Preferred amino- and carboxy-termini of fragments oranalogs occur near boundaries of functional domains. Structural andfunctional domains can be identified by comparison of the nucleotideand/or amino acid sequence data to public or proprietary sequencedatabases. Preferably, computerized comparison methods are used toidentify sequence motifs or predicted protein conformation domains thatoccur in other proteins of known structure and/or function. Methods toidentify protein sequences that fold into a known three-dimensionalstructure are known. Bowie et al. Science 253:164 (1991). Thus, theforegoing examples demonstrate that those of skill in the art canrecognize sequence motifs and structural conformations that may be usedto define structural and functional domains in accordance with theinvention.

Preferred amino acid substitutions are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinities, and (4) confer or modify other physicochemical orfunctional properties of such analogs. Analogs can include variousmuteins of a sequence other than the naturally-occurring peptidesequence. For example, single or multiple amino acid substitutions(preferably conservative amino acid substitutions) may be made in thenaturally-occurring sequence (preferably in the portion of thepolypeptide outside the domain(s) forming intermolecular contacts. Aconservative amino acid substitution should not substantially change thestructural characteristics of the parent sequence (e.g., a replacementamino acid should not tend to break a helix that occurs in the parentsequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles (Creighton, Ed., W. H. Freeman andCompany, New York (1984)); Introduction to Protein Structure (C. Brandenand J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); andThornton et at. Nature 354:105 (1991).

The term “polypeptide fragment” as used herein refers to a polypeptidethat has an amino terminal and/or carboxy-terminal deletion, but wherethe remaining amino acid sequence is identical to the correspondingpositions in the naturally-occurring sequence deduced, for example, froma full length cDNA sequence. Fragments typically are at least 5, 6, 8 or10 amino acids long, preferably at least 14 amino acids long′ morepreferably at least 20 amino acids long, usually at least 50 amino acidslong, and even more preferably at least 70 amino acids long. The term“analog” as used herein refers to polypeptides which are comprised of asegment of at least 25 amino acids that has substantial identity to aportion of a deduced amino acid sequence and which has specific bindingto TLR4/MD2 complex or TLR4 alone, under suitable binding conditions.Typically, polypeptide analogs comprise a conservative amino acidsubstitution (or addition or deletion) with respect to thenaturally-occurring sequence. Analogs typically are at least 20 aminoacids long, preferably at least 50 amino acids long or longer, and canoften be as long as a full-length naturally-occurring polypeptide.

Peptide analogs are commonly used in the pharmaceutical industry asnon-peptide drugs with properties analogous to those of the templatepeptide. These types of non-peptide compound are termed “peptidemimetics” or “peptidomimetics”. Fauchere, J. Adv. Drug Res. 15:29(1986), Veber and Freidinger TINS p. 392 (1985); and Evans et al. J.Med. Chem. 30:1229 (1987). Such compounds are often developed with theaid of computerized molecular modeling. Peptide mimetics that arestructurally similar to therapeutically useful peptides may be used toproduce an equivalent therapeutic or prophylactic effect. Generally,peptidomimetics are structurally similar to a paradigm polypeptide(i.e., a polypeptide that has a biochemical property or pharmacologicalactivity), such as human antibody, but have one or more peptide linkagesoptionally replaced by a linkage selected from the group consisting of:—CH₂NH—, —CH₂S—, —CH₂—CH₂—, —CH═CH—(cis and trans), —COCH₂—, CH(OH)CH₂—,and —CH₂SO—, by methods well known in the art. Systematic substitutionof one or more amino acids of a consensus sequence with a D-amino acidof the same type (e.g., D-lysine in place of L-lysine) may be used togenerate more stable peptides. In addition, constrained peptidescomprising a consensus sequence or a substantially identical consensussequence variation may be generated by methods known in the art (Rizoand Gierasch Ann. Rev. Biochem. 61:387 (1992)); for example, by addinginternal cysteine residues capable of forming intramolecular disulfidebridges which cyclize the peptide.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule, or an extract madefrom biological materials.

As used herein, the terms “label” or “labeled” refers to incorporationof a detectable marker, e.g., by incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotinyl moieties that can bedetected by marked avidin (e.g., streptavidin containing a fluorescentmarker or enzymatic activity that can be detected by optical orcalorimetric methods). In certain situations, the label or marker canalso be therapeutic. Various methods of labeling polypeptides andglycoproteins are known in the art and may be used. Examples of labelsfor polypeptides include, but are not limited to, the following:radioisotopes or radionuclides (e.g.,³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc,¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent labels (e.g., FITC, rhodamine,lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase,p-galactosidase, luciferase, alkaline phosphatase), chemiluminescent,biotinyl groups, predetermined polypeptide epitopes recognized by asecondary reporter (e.g., leucine zipper pair sequences, binding sitesfor secondary antibodies, metal binding domains, epitope tags). In someembodiments, labels are attached by spacer arms of various lengths toreduce potential steric hindrance. The term “pharmaceutical agent ordrug” as used herein refers to a chemical compound or compositioncapable of inducing a desired therapeutic effect when properlyadministered to a patient.

Other chemistry terms herein are used according to conventional usage inthe art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms(Parker, S., Ed., McGraw-Hill, San Francisco (1985)).

The term “antineoplastic agent” is used herein to refer to agents thathave the functional property of inhibiting a development or progressionof a neoplasm in a human, particularly a malignant (cancerous) lesion,such as a carcinoma, sarcoma, lymphoma, or leukemia. Inhibition ofmetastasis is frequently a property of antineoplastic agents.

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition), and preferably asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50 percent (on a molar basis) of allmacromolecular species present.

Generally, a substantially pure composition will comprise more thanabout 80 percent of all macromolecular species present in thecomposition, more preferably more than about 85%, 90%, 95%, and 99%.Most preferably, the object species is purified to essential homogeneity(contaminant species cannot be detected in the composition byconventional detection methods) wherein the composition consistsessentially of a single macromolecular species.

The term patient includes human and veterinary subjects.

Antibodies

Monoclonal antibodies of the invention (e.g., murine monoclonal orhumanized antibodies) have the ability to inhibit LPS-inducedproinflammatory cytokine production. Inhibition is determined, forexample, in the human whole blood and huTLR4/MD2 transfected HEK 293cellular assays described herein. Exemplary monoclonal antibodiesinclude, for example, the antibodies referred to herein as “mu18H10”,“hu18H10”, “mu16G7”, “mu15C1”, “hu15C1”, “mu7E3” and “hu7E3”. Themu18H10 and hu18H10 antibodies recognize the TLR4/MD-2 complex, but donot recognize an MD-2 protein when not complexed with TLR4. The mu16G7,mu15C1, hu15C1, mu7E3 and hu7E3 monoclonal antibodies recognize theTLR4/MD-2 complex. mu15C1, hu15C1 and 16G7 also recognize TLR4 when notcomplexed with MD-2.

Also included in the invention are antibodies that bind to the sameepitope as the antibodies described herein. For example, antibodies ofthe invention immunospecifically bind a TLR4/MD-2 complex, wherein theantibody binds to an epitope that includes one or more amino acidresidues on human TLR4 between residues 289 and 375 of SEQ ID NO:54.Antibodies of the invention immunospecifically bind the TLR4/MD2complex, wherein the antibody binds to an epitope on human MD-2 betweenresidues 19 and 57 of SEQ ID NO:44. Those skilled in the art willrecognize that it is possible to determine, without undueexperimentation, if a monoclonal antibody (e.g., a murine monoclonal orhumanized antibody) has the same specificity as a monoclonal antibody ofthe invention (e.g., mu18H10, hu18H10, mu16G7, mu15C1, hu15C1, mu7E3and/or hu7E3) by ascertaining whether the former prevents the latterfrom binding to the TLR4/MD-2 complex or to TLR4 when not complexed toMD-2. If the monoclonal antibody being tested competes with themonoclonal antibody of the invention, as shown by a decrease in bindingby the monoclonal antibody of the invention, then the two monoclonalantibodies bind to the same, or a closely related, epitope. Analternative method for determining whether a monoclonal antibody has thespecificity of monoclonal antibody of the invention is to pre-incubatethe monoclonal antibody of the invention with the TLR4/MD-2 complex or asoluble TLR4 protein (with which it is normally reactive), and then addthe monoclonal antibody being tested to determine if the monoclonalantibody being tested is inhibited in its ability to bind the TLR4/MD-2complex or to bind TLR4 and TLR4 complexed with MD-2. If the monoclonalantibody being tested is inhibited then, in all likelihood, it has thesame, or functionally equivalent, epitopic specificity as the monoclonalantibody of the invention. Screening of monoclonal antibodies of theinvention, can be also carried out by measuring LPS-induced IL-8production and determining whether the test monoclonal antibody is ableto neutralize LPS-induced IL-8 production.

Various procedures known within the art may be used for the productionof polyclonal or monoclonal antibodies directed against the TLR4/MD-2complex, or to TLR4 when not complexed to MD-2, or against derivatives,fragments, analogs homologs or orthologs thereof. (See, for example,Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., incorporated hereinby reference).

Antibodies are purified by well-known techniques, such as affinitychromatography using protein A or protein G, which provide primarily theIgG fraction of immune serum. Subsequently, or alternatively, thespecific antigen which is the target of the immunoglobulin sought, or anepitope thereof, may be immobilized on a column to purify the immunespecific antibody by immunoaffinity chromatography. Purification ofimmunoglobulins is discussed, for example, by D. Wilkinson (TheScientist, published by The Scientist, Inc., Philadelphia Pa., Vol. 14,No. 8 (Apr. 17, 2000), pp. 25-28).

The antibodies of the invention (e.g., hu18H10, 16G7, hu15C1 and hu7E3)are monoclonal antibodies. Monoclonal antibodies that neutralizeLPS-signaling that is mediated by the TLR4/MD-2 complex are generated,e.g., by immunizing BALB/c mice with combinations of cell transfectantsexpressing high levels of TLR4 and MD-2 on their surface and arecombinant soluble chimeric protein comprising both TLR4 and MD-2tethered by a flexible linker sequence. Hybridomas resulting frommyeloma/B cell fusions are then screened for reactivity to thisTLR4/MD-2 complex.

Monoclonal antibodies are prepared, for example, using hybridomamethods, such as those described by Kohler and Milstein, Nature, 256:495(1975). In a hybridoma method, a mouse, hamster, or other appropriatehost animal, is typically immunized with an immunizing agent to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the immunizing agent. Alternatively, thelymphocytes can be immunized in vitro.

The immunizing agent will typically include the protein antigen, afragment thereof or a fusion protein thereof. Generally, eitherperipheral blood lymphocytes are used if cells of human origin aredesired, or spleen cells or lymph node cells are used if non-humanmammalian sources are desired. The lymphocytes are then fused with animmortalized cell line using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, Academic Press, (1986) pp. 59-103).Immortalized cell lines are usually transformed mammalian cells,particularly myeloma cells of rodent, bovine and human origin. Usually,rat or mouse myeloma cell lines are employed. The hybridoma cells can becultured in a suitable culture medium that preferably contains one ormore substances that inhibit the growth or survival of the unfused,immortalized cells. For example, if the parental cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (“HAT medium”), which substances prevent thegrowth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of monoclonalantibodies. (See Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63)).

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against theantigen. Preferably, the binding specificity of monoclonal antibodiesproduced by the hybridoma cells is determined by immunoprecipitation orby an in vitro binding assay, such as radioimmunoassay (RIA) orenzyme-linked immunoabsorbent assay (ELISA). Such techniques and assaysare known in the art. The binding affinity of the monoclonal antibodycan, for example, be determined by the Scatchard analysis of Munson andPollard, Anal. Biochem., 107:220 (1980). Moreover, in therapeuticapplications of monoclonal antibodies, it is important to identifyantibodies having a high degree of specificity and a high bindingaffinity for the target antigen.

After the desired hybridoma cells are identified, the clones can besubcloned by limiting dilution procedures and grown by standard methods.(See Goding, Monoclonal Antibodies: Principles and Practice, AcademicPress, (1986) pp. 59-103). Suitable culture media for this purposeinclude, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640medium. Alternatively, the hybridoma cells can be grown in vivo asascites in a mammal.

The monoclonal antibodies secreted by the subclones can be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

Monoclonal antibodies can also be made by recombinant DNA methods, suchas those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA can be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also can be modified, for example, bysubstituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences (see U.S.Pat. No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or bycovalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide. Such anon-immunoglobulin polypeptide can be substituted for the constantdomains of an antibody of the invention, or can be substituted for thevariable domains of one antigen-combining site of an antibody of theinvention to create a chimeric bivalent antibody.

Monoclonal antibodies of the invention include humanized antibodies orhuman antibodies. These antibodies are suitable for administration tohumans without engendering an immune response by the human against theadministered immunoglobulin. Humanized forms of antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences of antibodies)that are principally comprised of the sequence of a humanimmunoglobulin, and contain minimal sequence derived from a non-humanimmunoglobulin. Humanization is performed, e.g., by following the methodof Winter and co-workers (Jones et al., Nature, 321:522-525 (1986);Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science,239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences forthe corresponding sequences of a human antibody. (See also U.S. Pat. No.5,225,539.) In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies also comprise, .e.g., residues which are foundneither in the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody includes substantially allof at least one, and typically two, variable domains, in which all orsubstantially all of the CDR regions correspond to those of a non-humanimmunoglobulin and all or substantially all of the framework regions arethose of a human immunoglobulin consensus sequence. The humanizedantibody optimally also includes at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin (Jones etal., 1986; Riechmann et al., 1988; and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)).

Fully human antibodies are antibody molecules in which the entiresequence of both the light chain and the heavy chain, including theCDRs, arise from human genes. Such antibodies are termed “humanantibodies”, or “fully human antibodies” herein. Monoclonal antibodiescan be prepared by using trioma technique; the human B-cell hybridomatechnique (see Kozbor, et al., 1983 Immunol Today 4: 72); and the EBVhybridoma technique to produce monoclonal antibodies (see Cole, et al.,1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc.,pp. 77-96). Monoclonal antibodies may be utilized and may be produced byusing human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA80: 2026-2030) or by transforming human B-cells with Epstein Barr Virusin vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCERTHERAPY, Alan R. Liss, Inc., pp. 77-96).

In addition, human antibodies can also be produced using additionaltechniques, including phage display libraries. (See Hoogenboom andWinter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol.,222:581 (1991)). Similarly, human antibodies can be made by introducinghuman immunoglobulin loci into transgenic animals, e.g., mice in whichthe endogenous immunoglobulin genes have been partially or completelyinactivated. Upon challenge, human antibody production is observed,which closely resembles that seen in humans in all respects, includinggene rearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al.,Bio/Technology 10, 779-783 (1992); Lonberg et al., Nature 368 856-859(1994); Morrison, Nature 368, 812-13 (1994); Fishwild et al, NatureBiotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826(1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995).

Human antibodies may additionally be produced using transgenic nonhumananimals which are modified so as to produce fully human antibodiesrather than the animal's endogenous antibodies in response to challengeby an antigen. (See PCT publication WO94/02602). The endogenous genesencoding the heavy and light immunoglobulin chains in the nonhuman hosthave been incapacitated, and active loci encoding human heavy and lightchain immunoglobulins are inserted into the host's genome. The humangenes are incorporated, for example, using yeast artificial chromosomescontaining the requisite human DNA segments. An animal which providesall the desired modifications is then obtained as progeny bycrossbreeding intermediate transgenic animals containing fewer than thefull complement of the modifications. An example of such a nonhumananimal is a mouse termed the Xenomouse™ as disclosed in PCT publicationsWO 96/33735 and WO 96/34096. This animal produces B cells which secretefully human immunoglobulins. The antibodies can be obtained directlyfrom the animal after immunization with an immunogen of interest, as,for example, a preparation of a polyclonal antibody, or alternativelyfrom immortalized B cells derived from the animal, such as hybridomasproducing monoclonal antibodies. Additionally, the genes encoding theimmunoglobulins with human variable regions can be recovered andexpressed to obtain the antibodies directly, or can be further modifiedto obtain analogs of antibodies such as, for example, single chain Fv(scFv) molecules.

An example of a method of producing a nonhuman host, exemplified as amouse, lacking expression of an endogenous immunoglobulin heavy chain isdisclosed in U.S. Pat. No. 5,939,598. It can be obtained by a method,which includes deleting the J segment genes from at least one endogenousheavy chain locus in an embryonic stem cell to prevent rearrangement ofthe locus and to prevent formation of a transcript of a rearrangedimmunoglobulin heavy chain locus, the deletion being effected by atargeting vector containing a gene encoding a selectable marker; andproducing from the embryonic stem cell a transgenic mouse whose somaticand germ cells contain the gene encoding the selectable marker.

One method for producing an antibody of interest, such as a humanantibody, is disclosed in U.S. Pat. No. 5,916,771. This method includesintroducing an expression vector that contains a nucleotide sequenceencoding a heavy chain into one mammalian host cell in culture,introducing an expression vector containing a nucleotide sequenceencoding a light chain into another mammalian host cell, and fusing thetwo cells to form a hybrid cell. The hybrid cell expresses an antibodycontaining the heavy chain and the light chain.

In a further improvement on this procedure, a method for identifying aclinically relevant epitope on an immunogen, and a correlative methodfor selecting an antibody that binds immunospecifically to the relevantepitope with high affinity, are disclosed in PCT publication WO99/53049.

The antibody can be expressed by a vector containing a DNA segmentencoding the single chain antibody described above.

These can include vectors, liposomes, naked DNA, adjuvant-assisted DNA.gene gun, catheters, etc. Vectors include chemical conjugates such asdescribed in WO 93/64701, which has targeting moiety (e.g. a ligand to acellular surface receptor), and a nucleic acid binding moiety (e.g.polylysine), viral vector (e.g. a DNA or RNA viral vector), fusionproteins such as described in PCT/US 95/02140 (WO 95/22618) which is afusion protein containing a target moiety (e.g. an antibody specific fora target cell) and a nucleic acid binding moiety (e.g. a protamine),plasmids, phage, etc. The vectors can be chromosomal, non-chromosomal orsynthetic.

Preferred vectors include viral vectors, fusion proteins and chemicalconjugates. Retroviral vectors include moloney murine leukemia viruses.DNA viral vectors are preferred. These vectors include pox vectors suchas orthopox or avipox vectors, herpesvirus vectors such as a herpessimplex I virus (HSV) vector (see Geller, A. I. et al., J. Neurochem,64:487 (1995); Lim, F., et al., in DNA Cloning: Mammalian Systems, D.Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, A. I.et al., Proc Natl. Acad. Sci.: U.S.A. 90:7603 (1993); Geller, A. I., etal., Proc Natl. Acad. Sci USA 87:1149 (1990), Adenovirus Vectors (seeLeGal LaSalle et al., Science, 259:988 (1993); Davidson, et al., Nat.Genet 3:219 (1993); Yang, et al., J. Virol. 69:2004 (1995) andAdeno-associated Virus Vectors (see Kaplitt, M. G., et al., Nat. Genet.8:148 (1994).

Pox viral vectors introduce the gene into the cells cytoplasm. Avipoxvirus vectors result in only a short term expression of the nucleicacid. Adenovirus vectors, adeno-associated virus vectors and herpessimplex virus (HSV) vectors are preferred for introducing the nucleicacid into neural cells. The adenovirus vector results in a shorter termexpression (about 2 months) than adeno-associated virus (about 4months), which in turn is shorter than HSV vectors. The particularvector chosen will depend upon the target cell and the condition beingtreated. The introduction can be by standard techniques, e.g. infection,transfection, transduction or transformation. Examples of modes of genetransfer include e.g., naked DNA, CaPO₄ precipitation, DEAE dextran,electroporation, protoplast fusion, lipofection, cell microinjection,and viral vectors.

The vector can be employed to target essentially any desired targetcell. For example, stereotaxic injection can be used to direct thevectors (e.g. adenovirus, HSV) to a desired location. Additionally, theparticles can be delivered by intracerebroventricular (icv) infusionusing a minipump infusion system, such as a SynchroMed Infusion System.A method based on bulk flow, termed convection, has also proveneffective at delivering large molecules to extended areas of the brainand may be useful in delivering the vector to the target cell. (See Boboet al., Proc. Natl. Acad. Sci. USA 91:2076-2080 (1994); Morrison et al.,Am. J. Physiol. 266:292-305 (1994)). Other methods that can be usedinclude catheters, intravenous, parenteral, intraperitoneal andsubcutaneous injection, and oral or other known routes ofadministration.

These vectors can be used to express large quantities of antibodies thatcan be used in a variety of ways. For example, to detect the presence ofthe TLR4/MD-2 complex and/or TLR4 in a sample. The antibody can also beused to try to bind to and disrupt TLR4/MD-2 complex-related signaling.

Techniques can be adapted for the production of single-chain antibodiesspecific to an antigenic protein of the invention (see e.g., U.S. Pat.No. 4,946,778). In addition, methods can be adapted for the constructionof Fab expression libraries (see e.g., Huse, et al., 1989 Science 246:1275-1281) to allow rapid and effective identification of monoclonalF_(ab) fragments with the desired specificity for a protein orderivatives, fragments, analogs or homologs thereof. Antibody fragmentsthat contain the idiotypes to a protein antigen may be produced bytechniques known in the art including, but not limited to: (i) anF_((ab′)2) fragment produced by pepsin digestion of an antibodymolecule; (ii) an F_(ab) fragment generated by reducing the disulfidebridges of an F_((ab′)2) fragment; (iii) an F_(ab) fragment generated bythe treatment of the antibody molecule with papain and a reducing agentand (iv) F_(v) fragments.

The invention also includes F_(v), F_(ab), F_(ab′) and F_((ab′)2)anti-TLR4/MD2 complex fragments or anti-TLR4 fragments, single chainanti-TLR4/MD2 or anti-TLR4 antibodies, bispecific anti-TLR4/MD2 oranti-TLR4 antibodies and heteroconjugate anti-TLR4/MD2 or anti-TLR4antibodies.

Bispecific antibodies are antibodies that have binding specificities forat least two different antigens. In the present case, one of the bindingspecificities is for the TLR4/MD2 complex and/or TLR4 when not complexedwith MD-2. The second binding target is any other antigen, andadvantageously is a cell-surface protein or receptor or receptorsubunit.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature, 305:537-539 (1983)). Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659(1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986).

According to another approach described in WO 96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers which are recovered from recombinant cellculture. The preferred interface comprises at least a part of the CH3region of an antibody constant domain. In this method, one or more smallamino acid side chains from the interface of the first antibody moleculeare replaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies can be prepared as full length antibodies orantibody fragments (e.g. F(ab′)₂ bispecific antibodies). Techniques forgenerating bispecific antibodies from antibody fragments have beendescribed in the literature. For example, bispecific antibodies can beprepared using chemical linkage. Brennan et al., Science 229:81 (1985)describe a procedure wherein intact antibodies are proteolyticallycleaved to generate F(ab′)₂ fragments. These fragments are reduced inthe presence of the dithiol complexing agent sodium arsenite tostabilize vicinal dithiols and prevent intermolecular disulfideformation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Additionally, Fab′ fragments can be directly recovered from E. coli andchemically coupled to form bispecific antibodies. Shalaby et al., J.Exp. Med. 175:217-225 (1992) describe the production of a fullyhumanized bispecific antibody F(ab′)₂ molecule. Each Fab′ fragment wasseparately secreted from E. coli and subjected to directed chemicalcoupling in vitro to form the bispecific antibody. The bispecificantibody thus formed was able to bind to cells overexpressing the ErbB2receptor and normal human T cells, as well as trigger the lytic activityof human cytotoxic lymphocytes against human breast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See, Gruber et al., J. Immunol. 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60(1991).

Exemplary bispecific antibodies can bind to two different epitopes, atleast one of which originates in the protein antigen of the invention.Alternatively, an anti-antigenic arm of an immunoglobulin molecule canbe combined with an arm which binds to a triggering molecule on aleukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, orB7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32)and FcγRIII (CD16) so as to focus cellular defense mechanisms to thecell expressing the particular antigen. Bispecific antibodies can alsobe used to direct cytotoxic agents to cells which express a particularantigen. These antibodies possess an antigen-binding arm and an armwhich binds a cytotoxic agent or a radionuclide chelator, such asEOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interestbinds the protein antigen described herein and further binds tissuefactor (TF).

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (see U.S. Pat. No.4,676,980), and for treatment of HIV infection (see WO 91/00360; WO92/200373; EP 03089). It is contemplated that the antibodies can beprepared in vitro using known methods in synthetic protein chemistry,including those involving crosslinking agents. For example, immunotoxinscan be constructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

It can be desirable to modify the antibody of the invention with respectto effector function, so as to enhance, e.g., the effectiveness of theantibody in treating diseases and disorders associated with aberrant LPSsignaling. For example, cysteine residue(s) can be introduced into theFc region, thereby allowing interchain disulfide bond formation in thisregion. The homodimeric antibody thus generated can have improvedinternalization capability and/or increased complement-mediated cellkilling and antibody-dependent cellular cytotoxicity (ADCC). (See Caronet al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J. Immunol., 148:2918-2922 (1992)). Alternatively, an antibody can be engineered that hasdual Fc regions and can thereby have enhanced complement lysis and ADCCcapabilities. (See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230(1989)).

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a toxin (e.g., an enzymaticallyactive toxin of bacterial, fungal, plant, or animal origin, or fragmentsthereof), or a radioactive isotope (i e., a radioconjugate).

Enzymatically active toxins and fragments thereof that can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, and the tricothecenes. A variety of radionuclides areavailable for the production of radioconjugated antibodies. Examplesinclude ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, and ¹⁸⁶Re.

Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. (See WO94/11026).

Those of ordinary skill in the art will recognize that a large varietyof possible moieties can be coupled to the resultant antibodies of theinvention. (See, for example, “Conjugate Vaccines”, Contributions toMicrobiology and Immunology, J. M. Cruse and R. E. Lewis, Jr (eds),Carger Press, New York, (1989), the entire contents of which areincorporated herein by reference).

Coupling may be accomplished by any chemical reaction that will bind thetwo molecules so long as the antibody and the other moiety retain theirrespective activities. This linkage can include many chemicalmechanisms, for instance covalent binding, affinity binding,intercalation, coordinate binding and complexation. The preferredbinding is, however, covalent binding. Covalent binding can be achievedeither by direct condensation of existing side chains or by theincorporation of external bridging molecules. Many bivalent orpolyvalent linking agents are useful in coupling protein molecules, suchas the antibodies of the present invention, to other molecules. Forexample, representative coupling agents can include organic compoundssuch as thioesters, carbodiimides, succinimide esters, diisocyanates,glutaraldehyde, diazobenzenes and hexamethylene diamines. This listingis not intended to be exhaustive of the various classes of couplingagents known in the art but, rather, is exemplary of the more commoncoupling agents. (See Killen and Lindstrom, Jour. Immun. 133:1335-2549(1984); Jansen et al., Immunological Reviews 62:185-216 (1982); andVitetta et al., Science 238:1098 (1987).

Preferred linkers are described in the literature. (See, for example,Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984) describing use ofMBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also, U.S. Pat.No. 5,030,719, describing use of halogenated acetyl hydrazide derivativecoupled to an antibody by way of an oligopeptide linker. Particularlypreferred linkers include: (i) EDC(1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride; (ii) SMPT(4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pridyl-dithio)-toluene(Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio)propionamido]hexanoate (Pierce Chem. Co., Cat#21651G); (iv) Sulfo-LC-SPDP (sulfosuccinimidyl 6[3-(2-pyridyldithio)-propianamide]hexanoate (Pierce Chem. Co. Cat.#2165-G); and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem.Co., Cat. #24510) conjugated to EDC.

The linkers described above contain components that have differentattributes, thus leading to conjugates with differing physio-chemicalproperties. For example, sulfo-NHS esters of alkyl carboxylates are morestable than sulfo-NHS esters of aromatic carboxylates. NHS-estercontaining linkers are less soluble than sulfo-NHS esters. Further, thelinker SMPT contains a sterically hindered disulfide bond, and can formconjugates with increased stability. Disulfide linkages, are in general,less stable than other linkages because the disulfide linkage is cleavedin vitro, resulting in less conjugate available. Sulfo-NHS, inparticular, can enhance the stability of carbodimide couplings.Carbodimide couplings (such as EDC) when used in conjunction withsulfo-NHS, forms esters that are more resistant to hydrolysis than thecarbodimide coupling reaction alone.

The antibodies disclosed herein can also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad.Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

Particularly useful liposomes can be generated by the reverse-phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol, and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem., 257: 286-288 (1982) via a disulfide-interchange reaction.

Use of Antibodies Against the TLR4/MD2 Complex and Antibodies AgainstTLR4

It will be appreciated that administration of therapeutic entities inaccordance with the invention will be administered with suitablecarriers, excipients, and other agents that are incorporated intoformulations to provide improved transfer, delivery, tolerance, and thelike. A multitude of appropriate formulations can be found in theformulary known to all pharmaceutical chemists: Remington'sPharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, Pa.(1975)), particularly Chapter 87 by Blaug, Seymour, therein. Theseformulations include, for example, powders, pastes, ointments, jellies,waxes, oils, lipids, lipid (cationic or anionic) containing vesicles(such as Lipofectin™), DNA conjugates, anhydrous absorption pastes,oil-in-water and water-in-oil emulsions, emulsions carbowax(polyethylene glycols of various molecular weights), semi-solid gels,and semi-solid mixtures containing carbowax. Any of the foregoingmixtures may be appropriate in treatments and therapies in accordancewith the present invention, provided that the active ingredient in theformulation is not inactivated by the formulation and the formulation isphysiologically compatible and tolerable with the route ofadministration See also Baldrick P. “Pharmaceutical excipientdevelopment: the need for preclinical guidance.” Regul. ToxicolPharmacol. 32(2):210-8 (2000), Wang W. “Lyophilization and developmentof solid protein pharmaceuticals.” Int. J. Pharm. 203(1-2):1-60 (2000),Charman W N “Lipids, lipophilic drugs, and oral drug delivery-someemerging concepts.” J Pharm Sci.89(8):967-78 (2000), Powell et al.“Compendium of excipients for parenteral formulations” PDA J Pharm SciTechnol. 52:238-311 (1998) and the citations therein for additionalinformation related to formulations, excipients and carriers well knownto pharmaceutical chemists.

Therapeutic formulations of the invention, which include a monoclonalantibody of the invention (e.g., a murine monoclonal or humanizedmonoclonal antibody), are used to treat or alleviate a symptomassociated with an immune-related disorder. The present invention alsoprovides methods of treating or alleviating a symptom associated with animmune-related disorder. A therapeutic regimen is carried out byidentifying a subject, e.g., a human patient suffering from (or at riskof developing) an immune-related disorder, using standard methods.

Antibodies of the invention, which are capable of inhibiting LPS-inducedproinflammatory cytokine production, are useful therapeutic tools in thetreatment of disorders, such as, for example, acute inflammation andsepsis induced by microbial products (e.g., LPS) and exacerbationsarising from this acute inflammation, such as, for example, chronicobstructive pulmonary disease and asthma (see O'Neill, Curr. Opin.Pharmacol. 3: 396-403 (2003), hereby incorporated by reference in itsentirety). Such antibodies are also useful in treating neurodegenerativeautoimmune diseases. (Lehnardt et al., Proc. Natl. Acad. Sci. USA 100:8514-8519(2003), hereby incorporated by reference in its entirety).

In addition, the antibodies of the invention are also useful astherapeutic reagents in the treatment of diseases, such as, for example,osteoarthritis, which are caused by mechanical stress, which, in turn,induces endogenous soluble “stress” factors that trigger TLR4.Endogenous soluble stress factor include e.g., Hsp60 (see Ohashi et al.,J. Immunol. 164: 558-561 (2000)) and fibronectin (see Okamura et al., J.Biol. Chem. 276: 10229-10233 (2001) and heparan sulphate, hyaluronan,gp96, β-Defensin-2 or surfactant protein A (see e.g., Johnson et al.,Crit. Rev. Immunol., 23(1-2):15-44 (2003), each of which is herebyincorporated by reference in its entirety). The antibodies of theinvention are also useful in the treatment of a variety of disordersassociated with mechanical stress, such as for example, mechanicalstress that is associated with subjects and patients placed onrespirators, ventilators and other respiratory-assist devices. Forexample, the antibodies of the invention are useful in the treatment ofventilator-induced lung injury (“VILI”), also referred to asventilation-associated lung injury (“VALI”).

Other disease areas in which inhibiting TLR4 function could bebeneficial include, for example, chronic inflammation (e.g., chronicinflammation associated with allergic conditions and asthma), autoimmunediseases (e.g., inflammatory bowel disorder) and atherosclerosis (seeO'Neill, Curr. Opin. Pharmacol. 3: 396-403 (2003), hereby incorporatedby reference in its entirety).

Symptoms associated with these immune-related disorders include, forexample, inflammation, fever, general malaise, fever, pain, oftenlocalized to the inflamed area, rapid pulse rate, joint pain or aches(arthralgia), rapid breathing or other abnormal breathing patterns,chills, confusion, disorientation, agitation, dizziness, cough, dyspnea,pulmonary infections, cardiac failure, respiratory failure, edema,weight gain, mucopurulent relapses, cachexia, wheezing, headache, andabdominal symptoms such as, for example, abdominal pain, diarrhea orconstipation.

Efficaciousness of treatment is determined in association with any knownmethod for diagnosing or treating the particular immune-relateddisorder. Alleviation of one or more symptoms of the immune-relateddisorder indicates that the antibody confers a clinical benefit.

Methods for the screening of antibodies that possess the desiredspecificity include, but are not limited to, enzyme linked immunosorbentassay (ELISA) and other immunologically mediated techniques known withinthe art.

Antibodies directed against the TLR4/MD-2 complex or to TLR4 when notcomplexed to MD-2 (or a fragment thereof) may be used in methods knownwithin the art relating to the localization and/or quantitation of theTLR4/MD-2 complex or TLR4 (e.g., for use in measuring levels of theTLR4/MD-2 complex or TLR4 within appropriate physiological samples, foruse in diagnostic methods, for use in imaging the protein, and thelike). In a given embodiment, antibodies specific to the TLR4/MD-2complex, or TLR4, or derivative, fragment, analog or homolog thereof,that contain the antibody derived antigen binding domain, are utilizedas pharmacologically active compounds (referred to hereinafter as“Therapeutics”).

An antibody specific for the TLR4/MD-2 complex or TLR4 can be used toisolate the TLR4/MD-2 complex or a TLR4 polypeptide by standardtechniques, such as immunoaffinity, chromatography orimmunoprecipitation. Antibodies directed against the TLR4/MD-2 complexor a TLR4 protein (or a fragment thereof) can be used diagnostically tomonitor protein levels in tissue as part of a clinical testingprocedure, e.g., to, for example, determine the efficacy of a giventreatment regimen. Detection can be facilitated by coupling (i.e.,physically linking) the antibody to a detectable substance. Examples ofdetectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,and radioactive materials. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

Antibodies of the invention, including polyclonal, monoclonal, humanizedand fully human antibodies, may be used as therapeutic agents. Suchagents will generally be employed to treat or prevent a disease orpathology associated with aberrant TLR4 signaling in a subject. Anantibody preparation, preferably one having high specificity and highaffinity for its target antigen, is administered to the subject and willgenerally have an effect due to its binding with the target.Administration of the antibody may abrogate or inhibit or interfere withthe signaling function of the target (e.g., the TLR4/MD-2 complex).Administration of the antibody may abrogate or inhibit or interfere withthe binding of the target (e.g., TLR4) with an endogenous ligand (e.g.,TLR4 or the MD-2 accessory protein) to which it naturally binds. Forexample, the antibody binds to the target and neutralizes LPS-inducedproinflammatory cytokine production.

A therapeutically effective amount of an antibody of the inventionrelates generally to the amount needed to achieve a therapeuticobjective. As noted above, this may be a binding interaction between theantibody and its target antigen that, in certain cases, interferes withthe functioning of the target. The amount required to be administeredwill furthermore depend on the binding affinity of the antibody for itsspecific antigen, and will also depend on the rate at which anadministered antibody is depleted from the free volume other subject towhich it is administered. Common ranges for therapeutically effectivedosing of an antibody or antibody fragment of the invention may be, byway of nonlimiting example, from about 0.1 mg/kg body weight to about 50mg/kg body weight. Common dosing frequencies may range, for example,from twice daily to once a week.

Antibodies specifically binding the TLR4/MD-2 complex or a TLR4 proteinor a fragment thereof of the invention can be administered for thetreatment of disorders associated with aberrant LPS signaling in theform of pharmaceutical compositions. Principles and considerationsinvolved in preparing such compositions, as well as guidance in thechoice of components are provided, for example, in Remington: TheScience And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al.,editors) Mack Pub. Co., Easton, Pa.: 1995; Drug Absorption Enhancement:Concepts, Possibilities, Limitations, And Trends, Harwood AcademicPublishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery(Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

Where antibody fragments are used, the smallest inhibitory fragment thatspecifically binds to the binding domain of the target protein ispreferred. For example, based upon the variable-region sequences of anantibody, peptide molecules can be designed that retain the ability tobind the target protein sequence. Such peptides can be synthesizedchemically and/or produced by recombinant DNA technology. (See, e.g.,Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)). Theformulation can also contain more than one active compound as necessaryfor the particular indication being treated, preferably those withcomplementary activities that do not adversely affect each other.Alternatively, or in addition, the composition can comprise an agentthat enhances its function, such as, for example, a cytotoxic agent,cytokine, chemotherapeutic agent, or growth-inhibitory agent. Suchmolecules are suitably present in combination in amounts that areeffective for the purpose intended.

The active ingredients can also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacrylate)microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules) or in macroemulsions.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations can be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods.

An antibody according to the invention can be used as an agent fordetecting the presence of the TLR4/MD-2 complex or a TLR4 protein (or aprotein fragment thereof) in a sample. In some embodiments, the antibodycontains a detectable label. Antibodies are polyclonal, or morepreferably, monoclonal. An intact antibody, or a fragment thereof (e.g.,F_(ab), scFv, or F_((ab)2)) is used. The term “labeled”, with regard tothe probe or antibody, is intended to encompass direct labeling of theprobe or antibody by coupling (i.e., physically linking) a detectablesubstance to the probe or antibody, as well as indirect labeling of theprobe or antibody by reactivity with another reagent that is directlylabeled. Examples of indirect labeling include detection of a primaryantibody using a fluorescently-labeled secondary antibody andend-labeling of a DNA probe with biotin such that it can be detectedwith fluorescently-labeled streptavidin. The term “biological sample” isintended to include tissues, cells and biological fluids isolated from asubject, as well as tissues, cells and fluids present within a subject.Included within the usage of the term “biological sample”, therefore, isblood and a fraction or component of blood including blood serum, bloodplasma, or lymph. That is, the detection method of the invention can beused to detect an analyte mRNA, protein, or genomic DNA in a biologicalsample in vitro as well as in vivo. For example, in vitro techniques fordetection of an analyte mRNA include Northern hybridizations and in situhybridizations. In vitro techniques for detection of an analyte proteininclude enzyme linked immunosorbent assays (ELISAs), Western blots,immunoprecipitations, and immunofluorescence. In vitro techniques fordetection of an analyte genomic DNA include Southern hybridizations.Procedures for conducting immunoassays are described, for example in“ELISA: Theory and Practice: Methods in Molecular Biology”, Vol. 42, J.R. Crowther (Ed.) Human Press, Totowa, N.J., 1995; “Immunoassay”, E.Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, Calif.,1996; and “Practice and Theory of Enzyme Immunoassays”, P. Tijssen,Elsevier Science Publishers, Amsterdam, 1985. Furthermore, in vivotechniques for detection of an analyte protein include introducing intoa subject a labeled anti-analyte protein antibody. For example, theantibody can be labeled with a radioactive marker whose presence andlocation in a subject can be detected by standard imaging techniques.

Chimeric Polypeptides

The chimeric peptides of the invention include a first and second domainoperably linked together. The first domain includes at least a portionof a toll-like receptor polypeptide, while the second domain includes atleast a portion of an MD accessory protein. The first and second domainscan occur in any order in the peptide, and the peptide can include oneor more of each domain. The chimeric protein comprises at least onebiologically active portion of a toll-like receptor polypeptide or MDaccessory protein. The chimeric peptide is soluble. By soluble is meantthe ability to dissolve in a fluid.

A “toll-like receptor polypeptide” refers to a polypeptide having anamino acid sequence corresponding to at least a portion of a toll-likereceptor polypeptide. A toll-like receptor polypeptide includes, forexample, TLRs 1-10 and RP105. The toll-like receptor polypeptide, and/ornucleic acids encoding the toll-like receptor polypeptide, can beconstructed using toll-like receptor polypeptide encoding sequences thatare known in the art and are described in, e.g. GenBank Accession Nos.(CAH72620; CAH72619; NP_(—)003254; NP_(—)003255; NP_(—)003259;NP_(—)006059; NP_(—)057646; NP_(—)003256; AAH33651; CAD99157; AAM23001;BAB43955; AAF05316; AAK26744; AAF78037; AAF78036; AAF78035; BAB19259;AAF64061; AAF60188; AAF89753; AAF07823; BAA78631; AAC34135; AAC34134;AAC34133; AAC34137) and are incorporated herein by reference in theirentirety. Within the chimeric protein the toll-like receptor polypeptidecan correspond to all or a portion of a toll-like receptor polypeptide.Preferably the toll-like receptor polypeptide includes the extracellularportion of the polypeptide.

An “MD accessory protein” refers to a polypeptide having an amino acidsequence corresponding to at least a portion of a MD accessory protein.The MD protein is, e.g., MD-1 or MD-2. The MD accessory protein, and/ornucleic acids encoding the MD accessory protein, can be constructedusing MD accessory protein encoding sequences that are known in the artand are described in, e.g. GenBank Accession Nos. GenBank Accession Nos.O95711 (MD-1); AAC98152 (MD-1); Q9Y6Y9 (MD-2); NP_(—)056179 (MD-2);AAH20690 (MD-2); and BAA78717 (MD-2). Exemplary MD accessory protein andnucleic acid sequences are is shown in FIGS. 32A, 32B, 33A and 33B.Within the chimeric protein the MD accessory protein can correspond toall or a portion of a MD accessory protein.

The chimeric protein may be linked to one or more additional moieties.For example, the chimeric protein may additionally be linked to a GSTfusion protein in which the glycoprotein Iba fusion protein sequencesare fused to the C-terminus of the GST (i.e., glutathione S-transferase)sequences. Such fusion proteins can facilitate the purification ofchimeric protein.

In another embodiment, the chimeric protein is includes a heterologoussignal sequence (i.e., a polypeptide sequence that is not present in apolypeptide encoded by a toll-like receptor polypeptide or MD accessoryprotein nucleic acid) at its N-terminus. For example, the nativetoll-like receptor polypeptide signal sequence can be removed andreplaced with a signal sequence from another protein. In certain hostcells (e.g., mammalian host cells), expression and/or secretion ofchimeric protein can be increased through use of a heterologous signalsequence.

An chimeric protein of the invention can be produced by standardrecombinant DNA techniques. For example, DNA fragments coding for thedifferent polypeptide sequences are ligated together in-frame inaccordance with conventional techniques, e.g., by employing blunt-endedor stagger-ended termini for ligation, restriction enzyme digestion toprovide for appropriate termini, filling-in of cohesive ends asappropriate, alkaline phosphatase treatment to avoid undesirablejoining, and enzymatic ligation. In another embodiment, the fusion genecan be synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers that give rise to complementaryoverhangs between two consecutive gene fragments that can subsequentlybe annealed and reamplified to generate a chimeric gene sequence (see,for example, Ausubel et al. (eds.) CURRENT PROTOCOLS IN MOLECULARBIOLOGY, John Wiley & Sons, 1992). Moreover, many expression vectors arecommercially available that encode a fusion moiety (e.g., an Fc regionof an immunoglobulin heavy chain). A glycoprotein Iba encoding nucleicacid can be cloned into such an expression vector such that the fusionmoiety is linked in-frame to the immunoglobulin protein.

Within the chimeric protein, the term “operatively linked” is intendedto indicate that the first and second polypeptides are chemically linked(most typically via a covalent bond such as a peptide bond) in a mannerthat allows for at least one function associated with the toll-likereceptor polypeptide and MD accessory protein. When used to refer tonucleic acids encoding the chimeric protein the term operatively linkedmeans that a nucleic acid encoding the toll-like receptor polypeptide orMD accessory protein are fused in-frame to each other. The MD accessoryprotein can be fused to the N-terminus or C-terminus of the toll-likereceptor polypeptide. Optionally, the toll-like receptor polypeptide andMD accessory protein are linked via a spacer arm. Spacer arms provideintramolecular flexibility or adjust intramolecular distances betweenconjugated moieties and thereby may help preserve biological activity. Aspacer arm may be in the form of a polypeptide moiety that includesspacer amino acids, e.g. proline, serine or glycine. Preferably thetoll-like receptor polypeptide and MD accessory protein are linked via aflexible glycine/serine linker. Alternatively, a spacer arm may be partof the cross-linking reagent, such as in “long-chain SPDP” (Pierce Chem.Co., Rockford, Ill., cat. No. 21651 H).

In other embodiments, the toll-like receptor polypeptide and the MDaccessory protein are linked by chemical coupling in any suitable mannerknown in the art. Many known chemical cross-linking methods arenon-specific, i e.; they do not direct the point of coupling to anyparticular site on the toll-like polypeptide or MD accessory protein. Asa result, use of non-specific cross-linking agents may attack functionalsites or sterically block active sites, rendering the conjugatedproteins biologically inactive.

One way to increasing coupling specificity is to directly chemicalcoupling to a functional group found only once or a few times in one orboth of the polypeptides to be cross-linked. For example, in manyproteins, cysteine, which is the only protein amino acid containing athiol group, occurs only a few times. Also, for example, if apolypeptide contains no lysine residues, a cross-linking reagentspecific for primary amines will be selective for the amino terminus ofthat polypeptide. Successful utilization of this approach to increasecoupling specificity requires that the polypeptide have the suitablyrare and reactive residues in areas of the molecule that may be alteredwithout loss of the molecule's biological activity.

Cysteine residues may be replaced when they occur in parts of apolypeptide sequence where their participation in a cross-linkingreaction would otherwise likely interfere with biological activity. Whena cysteine residue is replaced, it is typically desirable to minimizeresulting changes in polypeptide folding. Changes in polypeptide foldingare minimized when the replacement is chemically and sterically similarto cysteine. For these reasons, serine is preferred as a replacement forcysteine. As demonstrated in the examples below, a cysteine residue maybe introduced into a polypeptide's amino acid sequence for cross-linkingpurposes. When a cysteine residue is introduced, introduction at or nearthe amino or carboxy terminus is preferred. Conventional methods areavailable for such amino acid sequence modifications, whether thepolypeptide of interest is produced by chemical synthesis or expressionof recombinant DNA.

Coupling of the two constituents can be accomplished via a coupling orconjugating agent. There are several intermolecular cross-linkingreagents which can be utilized, See for example, Means and Feeney,CHEMICAL MODIFICATION OF PROTEINS, Holden-Day, 1974, pp. 39-43. Amongthese reagents are, for example, J-succinimidyl3-(2-pyridyldithio)propionate (SPDP) or N,N′-(1,3-phenylene)bismaleimide(both of which are highly specific for sulfhydryl groups and formirreversible linkages); N,N′-ethylene-bis-(iodoacetamide) or other suchreagent having 6 to 11 carbon methylene bridges (which relativelyspecific for sulfhydryl groups); and 1,5-difluoro-2,4-dinitrobenzene(which forms irreversible linkages with amino and tyrosine groups).Other cross-linking reagents useful for this purpose include:p,p′-difluoro-m,m′-dinitrodiphenylsulfone (which forms irreversiblecross-linkages with amino and phenolic groups); dimethyl adipimidate(which is specific for amino groups); phenol-1,4-disulfonylchloride(which reacts principally with amino groups); hexamethylenediisocyanateor diisothiocyanate, or azophenyl-p-diisocyanate (which reactsprincipally with amino groups); glutaraldehyde (which reacts withseveral different side chains) and disdiazobenzidine (which reactsprimarily with tyrosine and histidine).

Cross-linking reagents may be homobifunctional, i.e., having twofunctional groups that undergo the same reaction. A preferredhomobifunctional cross-linking reagent is bismaleimidohexane (“BMH”).BMH contains two maleimide functional groups, which react specificallywith sulfhydryl-containing compounds under mild conditions (pH 6.5-7.7).The two maleimide groups are connected by a hydrocarbon chain.Therefore, BMH is useful for irreversible cross-linking of polypeptidesthat contain cysteine residues.

Cross-linking reagents may also be heterobifunctional.Heterobifunctional cross-linking agents have two different functionalgroups, for example an amine-reactive group and a thiol-reactive group,that will cross-link two proteins having free amines and thiols,respectively. Examples of heterobifunctional cross-linking agents aresuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (“SMCC”),m-maleimidobenzoyl-N-hydroxysuccinimide ester (“MBS”), and succinimide4-(p-maleimidophenyl)butyrate (“SMPB”), an extended chain analog of MBS.The succinimidyl group of these cross-linkers reacts with a primaryamine, and the thiol-reactive maleimide forms a covalent bond with thethiol of a cysteine residue.

Cross-linking reagents often have low solubility in water. A hydrophilicmoiety, such as a sulfonate group, may be added to the cross-linkingreagent to improve its water solubility. Sulfo-MBS and sulfo-SMCC areexamples of cross-linking reagents modified for water solubility.

Many cross-linking reagents yield a conjugate that is essentiallynon-cleavable under cellular conditions. However, some cross-linkingreagents contain a covalent bond, such as a disulfide, that is cleavableunder cellular conditions. For example, Traut's reagent,dithiobis(succinimidylpropionate) (“DSP”), and N-succinimidyl3-(2-pyridyldithio)propionate (“SPDP”) are well-known cleavablecross-linkers. The use of a cleavable cross-linking reagent permits thecargo moiety to separate from the transport polypeptide after deliveryinto the target cell. Direct disulfide linkage may also be useful.

Numerous cross-linking reagents, including the ones discussed above, arecommercially available. Detailed instructions for their use are readilyavailable from the commercial suppliers. A general reference on proteincross-linking and conjugate preparation is: Wong, CHEMISTRY OF PROTEINCONJUGATION AND CROSS-LINKING, CRC Press (1991).

Also included in the invention are derivatives, fragments, homologs,analogs and variants of the chimeric peptides and nucleic acids encodingthese peptides. For nucleic acids, derivatives, fragments, and analogsprovided herein are defined as sequences of at least 6 (contiguous)nucleic acids, and which have a length sufficient to allow for specifichybridization. For amino acids, derivatives, fragments, and analogsprovided herein are defined as sequences of at least 4 (contiguous)amino acids, a length sufficient to allow for specific recognition of anepitope.

The length of the fragments are less than the length of thecorresponding full-length nucleic acid or polypeptide from which thechimeric peptide, or nucleic acid encoding same, is derived. Derivativesand analogs may be full length or other than full length, if thederivative or analog contains a modified nucleic acid or amino acid.Derivatives or analogs of the chimeric peptides include, e.g., moleculesincluding regions that are substantially homologous to the peptides, invarious embodiments, by at least about 30%, 50%, 70%, 80%, or 95%, 98%,or even 99%, identity over an amino acid sequence of identical size orwhen compared to an aligned sequence in which the alignment is done by acomputer homology program known in the art. For example sequenceidentity can be measured using sequence analysis software (SequenceAnalysis Software Package of the Genetics Computer Group, University ofWisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis.53705), with the default parameters therein.

Where a particular polypeptide is said to have a specific percentidentity to a reference polypeptide of a defined length, the percentidentity is relative to the reference peptide. Thus, a peptide that is50% identical to a reference polypeptide that is 100 amino acids longcan be a 50 amino acid polypeptide that is completely identical to a 50amino acid long portion of the reference polypeptide. It might also be a100 amino acid long polypeptide, which is 50% identical to the referencepolypeptide over its entire length. Of course, other polypeptides willmeet the same criteria.

Pharmaceutical Compositions

The antibodies or soluble chimeric polypeptides of the invention (alsoreferred to herein as “active compounds”), and derivatives, fragments,analogs and homologs thereof, can be incorporated into pharmaceuticalcompositions suitable for administration. Such compositions typicallycomprise the antibody or soluble chimeric polypeptide and apharmaceutically acceptable carrier. As used herein, the term“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. Suitable carriers aredescribed in the most recent edition of Remington's PharmaceuticalSciences, a standard reference text in the field, which is incorporatedherein by reference. Preferred examples of such carriers or diluentsinclude, but are not limited to, water, saline, ringer's solutions,dextrose solution, and 5% human serum albumin. Liposomes and non-aqueousvehicles such as fixed oils may also be used. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid(EDTA); buffers such as acetates, citrates or phosphates, and agents forthe adjustment of tonicity such as sodium chloride or dextrose. The pHcan be adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™. (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Screening Methods

The invention provides methods (also referred to herein as “screeningassays”) for identifying modulators, i.e., candidate or test compoundsor agents (e.g., peptides, peptidomimetics, small molecules or otherdrugs) that modulate or otherwise interfere with the binding of TLR4 tothe MD-2 accessory protein, or candidate or test compounds or agentsthat modulate or otherwise interfere with the signaling function of TLR4and/or the TLR4/MD-2 complex. Also provided are methods of identifyingcompounds useful to treat disorders associated with aberrantLPS-signaling. The invention also includes compounds identified in thescreening assays described herein.

In one embodiment, the invention provides assays for screening candidateor test compounds which modulate the signaling function of the TLR4/MD-2complex and/or the interaction between TLR4 and MD-2. The test compoundsof the invention can be obtained using any of the numerous approaches incombinatorial library methods known in the art, including: biologicallibraries; spatially addressable parallel solid phase or solution phaselibraries; synthetic library methods requiring deconvolution; the“one-bead one-compound” library method; and synthetic library methodsusing affinity chromatography selection. The biological library approachis limited to peptide libraries, while the other four approaches areapplicable to peptide, non-peptide oligomer or small molecule librariesof compounds. (See, e.g., Lam, 1997. Anticancer Drug Design 12: 145).

A “small molecule” as used herein, is meant to refer to a compositionthat has a molecular weight of less than about 5 kD and most preferablyless than about 4 kD. Small molecules can be, e.g., nucleic acids,peptides, polypeptides, peptidomimetics, carbohydrates, lipids or otherorganic or inorganic molecules. Libraries of chemical and/or biologicalmixtures, such as fungal, bacterial, or algal extracts, are known in theart and can be screened with any of the assays of the invention.

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt, et al., 1993. Proc. Natl.Acad. Sci. U.S.A. 90: 6909; Erb, et al., 1994. Proc. Natl. Acad. Sci.U.S.A. 91: 11422; Zuckermann, et al., 1994. J. Med. Chem. 37: 2678; Cho,et al., 1993. Science 261: 1303; Carrell, et al., 1994. Angew. Chem.Int. Ed. Engl. 33: 2059; Carell, et al., 1994. Angew. Chem. Int. Ed.Engl. 33: 2061; and Gallop, et al., 1994. J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (see e.g., Houghten,1992. Biotechniques 13: 412-421), or on beads (see Lam, 1991. Nature354: 82-84), on chips (see Fodor, 1993. Nature 364: 555-556), bacteria(see U.S. Pat. No. 5,223,409), spores (see U.S. Pat. No. 5,233,409),plasmids (see Cull, et al., 1992. Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage (see Scott and Smith, 1990. Science 249: 386-390;Devlin, 1990. Science 249: 404-406; Cwirla, et al., 1990. Proc. Natl.Acad. Sci. U.S.A. 87: 6378-6382; Felici, 1991. J. Mol. Biol. 222:301-310; and U.S. Pat. No. 5,233,409.).

In one embodiment, a candidate compound is introduced to anantibody-antigen complex and determining whether the candidate compounddisrupts the antibody-antigen complex, wherein a disruption of thiscomplex indicates that the candidate compound modulates the signalingfunction of the TLR4/MD-2 complex and/or the interaction between TLR4and MD-2. For example, the antibody is monoclonal antibody mu18H10,hu18H10 and the antigen is the TLR4/MD-2 complex. Alternatively, themonoclonal antibody is 16G7, mu15C1, hu15C1, mu7E3 or hu7E3 and theantigen is the TLR4/MD-2 complex or TLR4.

In another embodiment, a TLR4/MD-2 complex is provided and exposed to atleast one neutralizing monoclonal antibody. Formation of anantibody-antigen complex is detected, and one or more candidatecompounds are introduced to the complex. If the antibody-antigen complexis disrupted following introduction of the one or more candidatecompounds, the candidate compounds is useful to treat disordersassociated with aberrant LPS-signaling.

In another embodiment, a soluble chimeric protein of the invention isprovided and exposed to at least one neutralizing monoclonal antibody.Formation of an antibody-antigen complex is detected, and one or morecandidate compounds are introduced to the complex. If theantibody-antigen complex is disrupted following introduction of the oneor more candidate compounds, the candidate compounds is useful to treatdisorders associated with aberrant LPS-signaling.

Determining the ability of the test compound to interfere with ordisrupt the antibody-antigen complex can be accomplished, for example,by coupling the test compound with a radioisotope or enzymatic labelsuch that binding of the test compound to the antigen orbiologically-active portion thereof can be determined by detecting thelabeled compound in a complex. For example, test compounds can belabeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, andthe radioisotope detected by direct counting of radioemission or byscintillation counting. Alternatively, test compounds can beenzymatically-labeled with, for example, horseradish peroxidase,alkaline phosphatase, or luciferase, and the enzymatic label detected bydetermination of conversion of an appropriate substrate to product.

In one embodiment, the assay comprises contacting an antibody-antigencomplex with a test compound, and determining the ability of the testcompound to interact with the antigen or otherwise disrupt the existingantibody-antigen complex. In this embodiment, determining the ability ofthe test compound to interact with the antigen and/or disrupt theantibody-antigen complex comprises determining the ability of the testcompound to preferentially bind to the antigen or a biologically-activeportion thereof, as compared to the antibody.

In another embodiment, the assay comprises contacting anantibody-antigen complex with a test compound and determining theability of the test compound to modulate the antibody-antigen complex.Determining the ability of the test compound to modulate theantibody-antigen complex can be accomplished, for example, bydetermining the ability of the antigen to bind to or interact with theantibody, in the presence of the test compound.

Those skilled in the art will recognize that, in any of the screeningmethods disclosed herein, the antibody may be a neutralizing antibody,such as monoclonal antibody hu18H10, hu15C1 and/or hu7E3, each of whichmodulates or otherwise interferes with LPS-induced proinflammatorycytokine production.

The screening methods disclosed herein may be performed as a cell-basedassay or as a cell-free assay. The cell-free assays of the invention areamenable to use of either the soluble form or the membrane-bound form ofthe TLR4 and/or TLR4 when complexed with MD-2, and fragments thereof. Inthe case of cell-free assays comprising the membrane-bound forms of TLR4and/or the TLR4/MD-2 complex, it may be desirable to utilize asolubilizing agent such that the membrane-bound form of the proteins aremaintained in solution. Examples of such solubilizing agents includenon-ionic detergents such as n-octylglucoside, n-dodecylglucoside,n-dodecylmaltoside, octanoyl-N-methylglucamide,decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®,Isotridecypoly(ethylene glycol ether)_(n),N-dodecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate,3-(3-cholamidopropyl)dimethylamminiol-1-propane sulfonate (CHAPS), or3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane sulfonate(CHAPSO).

In more than one embodiment, it may be desirable to immobilize eitherthe antibody or the antigen to facilitate separation of complexed fromuncomplexed forms of one or both following introduction of the candidatecompound, as well as to accommodate automation of the assay. Observationof the antibody-antigen complex in the presence and absence of acandidate compound, can be accomplished in any vessel suitable forcontaining the reactants. Examples of such vessels include microtiterplates, test tubes, and micro-centrifuge tubes. In one embodiment, afusion protein can be provided that adds a domain that allows one orboth of the proteins to be bound to a matrix. For example, GST-antibodyfusion proteins or GST-antigen fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtiter plates, that are then combined withthe test compound, and the mixture is incubated under conditionsconducive to complex formation (e.g., at physiological conditions forsalt and pH). Following incubation, the beads or microtiter plate wellsare washed to remove any unbound components, the matrix immobilized inthe case of beads, complex determined either directly or indirectly.Alternatively, the complexes can be dissociated from the matrix, and thelevel of antibody-antigen complex formation can be determined usingstandard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either theantibody (e.g. hu18H10, hu15C1, and/or hu7E3) or the antigen (e.g. theTLR4/MD-2 complex and/or a TLR4 protein) can be immobilized utilizingconjugation of biotin and streptavidin. Biotinylated antibody or antigenmolecules can be prepared from biotin-NHS (N-hydroxy-succinimide) usingtechniques well-known within the art (e.g., biotinylation kit, PierceChemicals, Rockford, Ill.), and immobilized in the wells ofstreptavidin-coated 96 well plates (Pierce Chemical). Alternatively,other antibodies reactive with the antibody or antigen of interest, butwhich do not interfere with the formation of the antibody-antigencomplex of interest, can be derivatized to the wells of the plate, andunbound antibody or antigen trapped in the wells by antibodyconjugation. Methods for detecting such complexes, in addition to thosedescribed above for the GST-immobilized complexes, includeimmunodetection of complexes using such other antibodies reactive withthe antibody or antigen.

The invention further pertains to novel agents identified by any of theaforementioned screening assays and uses thereof for treatments asdescribed herein.

Diagnostic Assays

Antibodies of the present invention can be detected by appropriateassays, e.g., conventional types of immunoassays. For example, asandwich assay can be performed in which the TLR4/MD-2 complex or a TLR4protein or fragment thereof is affixed to a solid phase. Incubation ismaintained for a sufficient period of time to allow the antibody in thesample to bind to the immobilized polypeptide on the solid phase. Afterthis first incubation, the solid phase is separated from the sample. Thesolid phase is washed to remove unbound materials and interferingsubstances such as non-specific proteins which may also be present inthe sample. The solid phase containing the antibody of interest (e.g.monoclonal antibody hu18H10, hu15C1 and/or hu7E3) bound to theimmobilized polypeptide is subsequently incubated with a second, labeledantibody or antibody bound to a coupling agent such as biotin or avidin.This second antibody may be another anti-TLR4/MD-2 complex antibody,another anti-TLR4 antibody or another antibody. Labels for antibodiesare well-known in the art and include radionuclides, enzymes (e.g.maleate dehydrogenase, horseradish peroxidase, glucose oxidase,catalase), fluors (fluorescein isothiocyanate, rhodamine, phycocyanin,fluorescarmine), biotin, and the like. The labeled antibodies areincubated with the solid and the label bound to the solid phase ismeasured. These and other immunoassays can be easily performed by thoseof ordinary skill in the art

An exemplary method for detecting the presence or absence of theTLR4/MD-2 complex or a TLR4 protein in a biological sample involvesobtaining a biological sample from a test subject and contacting thebiological sample with a labeled monoclonal antibody according to theinvention such that the presence of TLR4/MD-2 complex or TLR4 isdetected in the biological sample.

As used herein, the term “labeled”, with regard to the probe orantibody, is intended to encompass direct labeling of the probe orantibody by coupling (i.e., physically linking) a detectable substanceto the probe or antibody, as well as indirect labeling of the probe orantibody by reactivity with another reagent that is directly labeled.Examples of indirect labeling include detection of a primary antibodyusing a fluorescently-labeled secondary antibody and end-labeling of aDNA probe with biotin such that it can be detected withfluorescently-labeled streptavidin. The term “biological sample” isintended to include tissues, cells and biological fluids isolated from asubject, as well as tissues, cells and fluids present within a subject.That is, the detection method of the invention can be used to detectTLR4/MD-2 complex or TLR4 in a biological sample in vitro as well as invivo. For example, in vitro techniques for detection of TLR4/MD-2complex or TLR4 include enzyme linked immunosorbent assays (ELISAs),Western blots, immunoprecipitations, and immunofluorescence.Furthermore, in vivo techniques for detection of TLR4/MD-2 complex orTLR4 include introducing into a subject a labeled anti-TLR4/MD-2 complexor anti-TLR4 antibody. For example, the antibody can be labeled with aradioactive marker whose presence and location in a subject can bedetected by standard imaging techniques.

In one embodiment, the biological sample contains protein molecules fromthe test subject. One preferred biological sample is a peripheral bloodleukocyte sample isolated by conventional means from a subject.

The invention also encompasses kits for detecting the presence ofTLR4/MD-2 complex or TLR4 in a biological sample. For example, the kitcan comprise: a labeled compound or agent capable of detecting TLR4/MD-2complex or TLR4, when not complexed with MD-2, (e.g., an anti- TLR4/MD-2complex monoclonal antibody or an anti-TLR4 monoclonal antibody) in abiological sample; means for determining the amount of TLR4/MD-2 complexor TLR4 in the sample; and means for comparing the amount of TLR4/MD-2complex or TLR4 in the sample with a standard. The compound or agent canbe packaged in a suitable container. The kit can further compriseinstructions for using the kit to detect TLR4/MD-2 complex or TLR4 in asample.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Materials and Methods for the Generation of Murine18H10 Monoclonal Antibody

A. Generation of Stable TLR4IMD-2 Transfectants

Stable TLR4/MD-2 transfectants were generated in CHO-K1 and HEK 293cells. For CHO-K1 cells, human TLR4 cDNA encoding an N-terminal c-mycepitope tag was cloned into pCDNA3.1(−)hygro (Invitrogen), and humanMD-2 cDNA encoding C-terminal c-Myc and Protein C epitope tags wascloned into pCDNA3 (Invitrogen). Both constructs were co-transfectedinto CHO cells using Fugene 6™ reagent (Roche), according to themanufacturer's guidelines. Antibiotic resistant cells were selected inculture medium containing 500 μg/ml G418 and 250μg/ml hygromycin B (bothfrom Invitrogen).

To select for cells expressing the TLR4/MD-2 complex, 1×10⁷ cells/mlwere incubated in 1× PBS supplemented with 1% BSA and 10 μg/mlanti-protein C monoclonal antibody (Roche). Cells were washed once andthen incubated in the same buffer with PE-conjugated goat anti-mouse IgG(H+L) antibody (1:200 dilution; Anwara). Cells were subsequentlyincubated with anti-PE microbeads (Miltenyi Biotec) and passed through aMidi MACS LS column. Cells retained on the column were eluted and placedback in culture with antibiotic selection. Rounds of sorting werecontinued until a uniformly positive population of cells expressing theTLR4/MD-2 complex was obtained.

B. Generation of Recombinant MD-2 and Chimeric TLR4/MD-2 Protein

To generate recombinant soluble MD-2, cDNA encoding the protein with Cterminal FLAG and 6×HIS tags for detection and purification purposes wascloned into pFASTBAC1 and subsequently inserted into bacmid DNA byhomologous recombination. Following generation of a viral stock, Sf9cells were superinfected. 48 hours later, the recombinant protein waspurified from infected cell supernatants using a NiNTA affinity matrix(Qiagen).

To generate the recombinant TLR4/MD-2 chimeric protein, cDNA encodingthe extracellular portion of human TLR4 linked to MD-2 via a glycineserine (GGGGS₃) linker was assembled using PCR. FLAG and 6×HIS tags wereincluded at the C-terminus of MD-2 for detection and purificationpurposes. The cDNA cassette was cloned into the baculovirus expressionvector pFASTBAC1 (Invitrogen) and subsequently inserted into bacmid DNAby homologous recombination. Following generation of a viral stock, Sf9cells were superinfected. 48 hours later, the recombinant fusion proteinwas purified from cell lysates using an anti-FLAG™ M2 MAb affinitymatrix (Sigma).

C. Immunization of Mice

8 week old female BALB/c mice (IFFA CREDO) were immunized with asubcutaneous injection (s.c.) of 10⁶ CHO cells/ml in RIBI adjuvant(Sigma) at days 0, 7 and 28 as previously described in Buell et al.,Blood 92: 3521-3528 (1998), hereby incorporated by reference in itsentirety.

D. Specific Serum Titrations

The mice were bled at days 0 and 14. TLR4/MD-2 specific antibody titerswere assessed in the sera by FACS analysis on TLR4/MD-2 transfected 293cells. Cells were incubated with mice sera at 1:250, 1:2500 and 1:25000dilutions, washed, incubated with APC-conjugated goat anti-mouse IgG(H+L) antibody (Molecular Probes) and analyzed on a FACScalibur (BectonDickenson) in the FL-4 channel.

E. B Cell/Myeloma Fusions

Mice having specific TLR4/MD-2 serum antibodies were “hyperboosted”subcutaneously (s.c.) with the chimeric TLR4/MD-2 fusion protein either3 or 4 days prior to fusion. Draining lymph nodes were obtained as asource of B cells for fusion with the mouse myeloma cell lineP3-X63-Ag8.653. B cell extraction and cellular fusions were performed aspreviously described in Buell et al., Blood 92: 3521-3528 (1998), herebyincorporated by reference in its entirety. Cells were plated at anapproximate concentration of 10⁴ myeloma cells/well and grown for 10-14days in culture medium supplemented with HAT (Sigma).

F. Hybridoma Screening

Supernatants from wells containing viable hybridoma cells were screenedon mock transfected cells vs. TLR4/MD-2 transfected myeloma cells forTLR4/MD-2 specificity by FACS analysis. Cells were then incubated withsupernatant and goat-anti mouse IgG (H+L) antibody (Molecular Probes).Cells were analyzed on a FACScalibur in the FL-4 channel.

G. Monoclonal Antibody Specificity by FACS

HEK 293 cells were plated in 6 well plates at a density of 2.5×10⁵cells/well in 2 ml culture medium containing 10% FBS. 16 hourspost-plating, cells were transfected with 0.75 μg of the appropriatevector(s) using Fugene™ reagent (Roche) according to the manufacturer'sguidelines. 48 hours post-transfection, cells were stained with theappropriate monoclonal antibody (as indicated in FIG. 4) and anAPC-coupled goat anti-mouse IgG (H+L) antibody (Molecular Probes) andanalyzed using the FACScalibur in the FL-4 channel.

H. Monoclonal Antibody Specificity by Direct ELISA

Recombinant soluble MD-2 with C terminal FLAG and histidine epitope tagswas coated at a concentration of 5 μg/ml in 50 μl PBS on ELISA plates(Nunc Maxisorp). Wells were blocked with 200 μl PBS 2% BSA andsubsequently incubated with the appropriate MAb at the indicatedconcentration in PBS 1% BSA. Following 3 wash steps with PBS 0.05% Tween20, 50 μl HRP conjugated goat anti-mouse IgG (H+L) at a 1:5000 dilutionwas added to the wells. Following a further wash step, binding wasrevealed using TMB substrate. Plates were read at a wavelength of 650nm.

I Monoclonal Antibody Specificity by Sandwich ELISA

For sample preparation, HEK 293 cells were transfected with theappropriate plasmid constructs using the Fugene 6™ transfection reagentas described in paragraph G above. 48 hours post-transfection, cellswere collected and cleared by centrifugation. Cells were subsequentlyincubated with biotinylated mu18H10 (10 μg/ml) and lysed in 20 mM TrispH 7.4, 150 mM NaCl, 1% NP40 containing COMPLETE™ protease inhibitors(Roche).

To perform the sandwich ELISA, Nunc maxisorp plate wells were coatedwith 50 μl of the anti-FLAG™ M2 MAb (Sigma) at a concentration of 5μg/ml in PBS. Wells were blocked with 200 μl PBS 2% BSA and subsequentlyincubated with 50 μl of the appropriate samples at the indicateddilution. Wells were washed three times with 200 μl PBS 0.05% Tween 20and incubated with 50 μl of the appropriate antibody (10 μg/ml forbiotinylated mu18H10 and 12D4, 1 μg/ml for the polyclonal anti-MD-2MAb). Following a wash step as above, wells were incubated with 50 μl ofthe appropriate detection antibody (HRP conjugated streptavidin at adilution of 1:1500 for the biotinylated MAbs and HRP conjugatedanti-rabbit IgG (H+L) at a dilution of 1:5000 for the polyclonal rabbitAb). Following a further wash step, binding was revealed using TMBsubstrate. Plates were read at a wavelength of 650 nm.

J. Cellular Assay 1

Monoclonal antibody was first purified from hybridoma cell supernatantusing protein G affinity chromatography.

TLR4/MD-2 transfected HEK 293 cells were plated in culture mediumcontaining 10% FBS at 5×10⁵ cells/ml in 96 well plates and left toadhere overnight. The culture medium was subsequently removed andreplaced with 100 μl culture medium containing 2% FBS and theappropriate monoclonal antibody at twice the desired final concentrationfor 30 minutes at 37° C. LPS (K12LD25, Sigma) was then added to thecells at a concentration of 30 ng/ml in 100 μl culture medium containing2% FBS. Cells were incubated at 37° C. for 16 hours and supernatantsharvested. IL-8 content was measured by sandwich ELISA using themonoclonal antibody pair 801E and M802B (Endogen).

K. Cellular Assay 2

Human whole blood was diluted 1:4 in RPMI (Sigma) and plated at 100μl/well in 96 well plates with the appropriate monoclonal antibody attwice the desired final concentration for 30 minutes at 37° C. LPS(K12LD25, Sigma), dosed at twice the desired final concentration, wassubsequently added in 100 μl RPMI containing 5 mg/ml HSA and incubatedfor 6 hours at 37° C. Plates were then centrifuged at 2000 rpm for 5minutes and the supernatant from each well was retained. IL-8concentrations were determined by sandwich ELISA using the monoclonalantibody pair 801E and M802B (Endogen), as described above.

L. 18H10 VH and VL Sequences

10⁷ hybridoma cells were harvested and washed once with PBS before beingresuspended in 1 ml Trizol™ reagent (Invitrogen). Total RNA wassubsequently extracted according to the manufacturer's guidelines. cDNAencoding the VH and VL from three independent subclones of the mu18H10hybridoma was generated by RT-PCR using oligonucleotide primers specificfor mouse leader sequences and constant domains (Jones and Bendig,Biotechnology, 9: 88-89 (1991)). Amplified products were cloned into thepGEM-T easy vector (Promega Corp.) and sequenced using the T7 and SP6primers.

The VH and VL cDNAs were subsequently cloned in mammalian expressionvectors containing the human IgG1 and human kappa constant regionsrespectively in order to express 7E3 as a chimeric MAb (“chimeric 7E3”).To produce recombinant chimeric MAb, HEK 293 cells were plated in 6 wellplates at a density of 2.5×10⁵ cells/well in 2 ml culture mediumcontaining 10% FBS. 16 hours post-plating, cells were transfected with0.75 μg of the appropriate vector(s) using Fugene™ reagent (Roche)according to the manufacturer's guidelines. 48 hours post-transfection,supernatant was harvested and antibody was purified using protein Gaffinity chromatography.

Example 2 Generation of mu18H10 MAbs Directed Against the HumanTLR4/MD-2 Complex

Mice immunized with CHO cells expressing surface TLR4/MD-2 weremonitored for specific serum titers. Those showing a response toTLR4/MD-2 were “hyperboosted” with recombinant TLR4/MD-2 chimericprotein. This strategy was chosen in order to ensure that the immunesystem was initially exposed to a conformational TLR4/MD-2 complex andminimize the response to non-specific CHO cellular antigens andsimultaneously maximizing the TLR4/MD-2-specific response uponhyperboosting. Screening by FACS of supernatants from hybridomasresulting from B cell/myeloma fusions was performed on mock transfectedvs. TLR4/MD-2 transfected CHO cells. Monoclonal antibody from oneparticular clone, referred to herein as mu18H10, demonstrated specificbinding to TLR4/MD-2 transfected CHO cells (FIG. 1). This antibody wasfound to have the IgG2b κ isotype, as determined by FACS using the mouseIg isotyping CBA kit (Beckton Dickenson).

Example 3 mu18H10 MAb Neutralization of LPS Activity on TLR4/MD-2Transfected HEK 293 Cells

LPS is known to have the ability to induce IL-8 production in HEK 293cells transfected with the TLR4/MD-2 complex. The ability of mu18H10 toinhibit this IL-8 induction was analyzed by pre-incubating cells withthe antibody for 30 minutes prior to LPS administration. FIG. 2 showsthat mu18H10 inhibited the effects of LPS on HEK 293 cells, even atconcentrations below 1 μg/ml.

Example 4 mu18H10 MAb Neutralization of LPS Activity on Human WholeBlood

The ability of mu18H10 to inhibit LPS-induced IL-8 production in humanwhole blood was tested. mu18H10 neutralizing activity was tested inblood from 3 different donors using a range of monoclonal antibodyconcentrations from 0.5 to 10 μg/ml. FIG. 3 demonstrates that mu18H10significantly reduced the level of IL-8 induced by LPS in all 3 donors,as compared to an isotype matched control. mu18H10 was found to be morepotent than a previously described α-TLR4 blocking monoclonal antibody(purchased from e-biosciences). These results indicate that theneutralizing epitope recognized by mu18H10 on transfected HEK 293 cellsis also exposed on the surface on cells in whole blood, and that mu18H10is potent enough to inhibit the activity of LPS in whole blood, even atconcentrations below 1 μg/ml.

Example 5 mu18H10 Specificity

In order to determine the specificity of the mu18H10 monoclonalantibody, the fact that mu18H10 does not recognize the rabbit orthologof the TLR4/MD-2 complex (previously cloned) was exploited. cDNAs foreither human or rabbit TLR4 with N-terminal FLAG™ epitope tag and eitherhuman or rabbit MD-2 with C-terminal c-Myc and protein C epitope tagswere transfected in HEK 293 cells in the following combinations: (1)human TLR4 and human MD-2; (2) rabbit TLR4 and rabbit MD-2; (3) humanTLR4 and rabbit MD-2; (4) rabbit TLR4 and human MD-2. FIG. 4 shows FACSanalysis of these cells following antibody staining, which revealed thatmu18H10 recognized cells expressing the human TLR4/MD-2 complex and acombination of human TLR4 and rabbit MD-2, but not the rabbit TLR4/MD-2complex nor a combination of rabbit TLR4 and human MD-2. These resultsindicate that the epitope recognized by mu18H10 is situated on humanMD-2 (FIG. 4).

Although mu18H10 shows specificity for MD-2, it was determined thatmu18H10 only recognizes MD-2 in the context of its interaction withTLR4. Using direct ELISA, no binding of mu18H10 to recombinant solubleMD-2 generated with the baculovirus expression system was detected (FIG.5 a). In addition, FIG. 5 b reveals that mu18H10 only bound to a complexof TLR4 and MD-2 as shown from co-transfected cell lysates, and did notrecognize either MD-2 alone in transfected cell lysates/supernatants orTLR4 alone in transfected cell lysates. These data indicate that mu18H10is specific for the TLR4/MD-2 complex and does not recognize eithercomponent of the complex separately.

Example 6 mu18H10 VH and VL Sequences

VH and VL sequences from the mu18H10 hybridoma clone were amplified fromtotal RNA by RT-PCR. Sequence analysis is shown in FIGS. 6A-6F.

The mu18H10 antibody includes a heavy chain variable region (SEQ IDNO:2, FIG. 6B) encoded by the nucleic acid sequence of SEQ ID NO:1 shownin FIG. 6A, and a light chain variable region (SEQ ID NO:7, FIG. 6E)encoded by the nucleic acid sequence of SEQ ID NO:6 shown in FIG. 6D.The amino acids encompassing the complementarity determining regions(CDR) as defined by Chothia et al. 1989, E. A. Kabat et al., 1991 arehighlighted in underlined and italicized text in FIGS. 6B and 6E andshown in FIGS. 6C and 6F. (See Chothia, C, et al., Nature 342:877-883(1989); Kabat, E A, et al., Sequences of Protein of immunologicalinterest, Fifth Edition, US Department of Health and Human Services, USGovernment Printing Office (1991)). The heavy chain CDRs of the mu18H10antibody have the following sequences: DSYIH (SEQ ID NO:3);WTDPENVNSIYDPRFQG (SEQ ID NO:4), and GYNGVYYAMDY (SEQ ID NO:5). Thelight chain CDRs of the mu18H10 antibody have the following sequences:SASSSVIYMH (SEQ ID NO:8); RTYNLAS (SEQ ID NO:9); and HQWSSFPYT (SEQ IDNO:10).

Example 7 Chimeric 18H10 Binds to hTLR4 hMD2 Transfected CHO Cells

In order to demonstrate the specificity of the cloned 18H10 VH and VLfor the hTLR4/MD-2 complex, FACS analysis was performed on hTLR4/MD-2transfected CHO cells using the chimeric 18H10 MAb (FIG. 6). Specificbinding of MAb at the indicated concentration was detected using anAPC-labeled goat-anti-human IgG (H+L) secondary antibody. An irrelevantisotype-matched human IgG1 MAb was used as a control.

Example 8 Chimeric 18H10 Inhibits LPS-Induced IL-8 Production in hTLR4hMD2 Transfected HEK 293 Cells

In order to demonstrate the neutralizing capacity of the cloned 18H10 VHand VL for LPS, the ability of 18H10 to inhibit LPS dependent IL-8induction of hTLR4/MD-2 transfected HEK 293 cells was tested (asdescribed above). FIG. 7 shows that chimeric 18H10 inhibited the effectsof LPS on HEK 293 cells in a manner very similar to that of the original18H10 mouse MAb.

Example 9 Materials and Methods for the Generation of mu16G7 MonoclonalAntibody

A. Generation of Stable TLR4/MD-2 Transfectants

Stable TLR4/MD-2 transfectants were generated in CHO-K1 and HEK 293cells. For CHO-K1 cells, human TLR4 cDNA encoding an N-terminal c-mycepitope tag was cloned into pCDNA3.1(−)hygro (Invitrogen), and humanMD-2 cDNA encoding C-terminal c-Myc and Protein C epitope tags wascloned into pCDNA3 (Invitrogen). Both constructs were co-transfectedinto CHO cells using Fugene 6™ reagent (Roche), according to themanufacturer's guidelines. Antibiotic resistant cells were selected inculture medium containing 500 μg/ml G418 and 250 μg/ml hygromycin B(both from Invitrogen).

For HEK 293 cells, human TLR4 cDNA encoding an N-terminal FLAG™ epitopetag was cloned into pCDNA3.1(−)hygro (Invitrogen), and human MD-2 cDNAencoding C-terminal FLAG™ and 6×Histidine epitope tags was cloned intopCDNA3 (Invitrogen). Both constructs were transfected into HEK 293cells, and antibiotic resistant cells were selected in culture mediumcontaining 500 μg/ml G418 and 250μg/ml hygromycin B (both fromInvitrogen), as described above.

To select for cells expressing the TLR4/MD-2 complex, 1×10⁷ cells/mlwere incubated in 1× PBPS supplemented with 1% BSA and either 10 μg/mlanti-protein C monoclonal antibody (for CHO cells; Roche) or anti-FLAGmonoclonal antibody (for 293 cells; Sigma). Cells were washed once andthen incubated in the same buffer with PE-conjugated goat anti-mouse IgG(H+L) antibody (1:200 dilution; Anwara). Cells were subsequentlyincubated with anti-PE microbeads (Miltenyi Biotec) and passed through aMidi MACS LS column. Cells retained on the column were eluted and placedback in culture with antibiotic selection. Rounds of sorting werecontinued until a uniformly positive population of cells expressing theTLR4/MD-2 complex was obtained.

B. Immunization of Mice

8 week old female BALB/c mice (IFFA CREDO) were immunized as describedabove in Example 1, subsection C.

C. Specific Serum Titrations

Mice sera titrations were performed as described above in Example 1,subsection D.

D. B Cell/Myeloma Fusions

Mice having specific TLR4/MD-2 serum antibodies were “hyperboosted”subcutaneously (s.c.) with TLR4/MD-2 transfected HEK 293 either 3 or 4days prior to fusion. Draining lymph nodes were obtained as a source ofB cells for fusion with the mouse myeloma cell line P3-X63-Ag8.653. Bcell extraction and cellular fusions were performed as previouslydescribed in Buell et al., Blood 92: 3521-3528 (1998), herebyincorporated by reference in its entirety. Cells were plated at anapproximate concentration of 10⁴ myeloma cells/well and grown for 10-14days in culture medium supplemented with HAT (Sigma).

E. Hybridoma Screening

Hybridomas were screened as described above in Example 1, subsection F.

F. Monoclonal Antibody Specificity

The specificity of the mu16G7 monoclonal antibody was determined asdescribed above in Example 1, subsection G.

G. Cellular Assay 1

Cellular Assay I was performed as described above in Example 1,subsection J.

H. Cellular Assay 2

Cellular Assay II was performed as described above in Example 1,subsection K.

I. 16G7 VH and VL Sequences

10⁷ hybridoma cells were harvested and washed once with PBS before beingresuspended in 1 ml Trizol™ reagent (Invitrogen). Total RNA wassubsequently extracted according to the manufacturer's guidelines. cDNAencoding the VH and VL from the mu16G7 clone was generated by RT-PCRwith the mouse ScFv module (Amersham Biosciences) according to themanufacturer's guidelines. Amplified products were cloned into thepGEM-T easy vector (Promega Corp.) and sequenced using the T7 and SP6primers.

Example 10 Generation of mu16G7 MAbs Directed Against the HumanTLR4/MD-2 Complex

Mice immunized with CHO cells expressing surface TLR4/MD-2 weremonitored for specific serum titers. Those showing a response toTLR4/MD-2 were “hyperboosted” with HEK 293 TLR4/MD-2 transfectants. Thisstrategy was chosen in order to minimize the response to non-specificCHO cellular antigens, while simultaneously maximizing theTLR4/MD-2-specific response. Screening by FACS of supernatants fromhybridomas resulting from B cell/myeloma fusions was performed on mocktransfected vs. TLR4/MD-2 transfected CHO cells. Monoclonal antibodyfrom a specific clone, referred to herein as mu16G7, demonstratedspecific binding to TLR4/MD-2 transfected CHO cells (FIG. 9). mu16G7 wasfound to have the IgG1 κ isotype, as determined by FACS using the mouseIg isotyping CBA kit (Beckton Dickenson).

Example 11 mu16G7 Neutralization of LPS Activity on TLR4/MD-2Transfected HEK 293 Cells

LPS is known to have ability to induce IL-8 production in HEK 293 cellstransfected with the TLR4/MD-2 complex. The ability of mu16G7 to inhibitthis IL-8 induction was analyzed by pre-incubating cells with eachantibody for 30 minutes prior to LPS administration. FIG. 10 shows thatmu16G7 inhibited the effects of LPS on HEK 293 cells, even atsub-microgram/ml concentrations.

Example 12 mu16G7 Neutralization of LPS Activity on Human Whole Blood

The ability of mu16G7 to inhibit LPS-induced IL-8 production in humanwhole blood was tested. mu16G7 neutralizing activity was tested in bloodfrom 3 different donors using a range of monoclonal antibodyconcentrations from 0.5 to 5 μg/ml. FIG. 11 demonstrates that mu16G7significantly reduced the level of IL-8 induced by LPS in all 3 donors,as compared to an isotype matched control. mu16G7 was found to be morepotent than a previously described α-TLR4 blocking monoclonal antibody(from e-biosciences). (See Shimazu et al. J. Exp. Med. 189: 1777-1782(1999)). In some cases, mu16G7 was found to be as potent as an α-CD14blocking monoclonal antibody that was also included in the study. (SeeKirkland et al. J. Biol. Chem. 268: 24818-24823(1993)). These resultsindicate that the neutralizing epitope recognized by mu16G7 ontransfected HEK 293 cells is also exposed on the surface on cells inwhole blood, and that mu16G7 is potent enough to inhibit the activity ofLPS in whole blood, even at concentrations below 1 μg/ml.

Example 13 mu16G7 Specificity

In order to determine the specificity of the mu16G7 monoclonal antibody,the fact that mu16G7 does not recognize the rabbit ortholog of theTLR4/MD-2 complex (previously cloned) was exploited. cDNAs for eitherrabbit or human TLR4 with N-terminal FLAG™ epitope tag and MD-2 withC-terminal c-Myc and protein C epitope tags were transfected in HEK 293cells in the following combinations: (1) rabbit TLR4 and rabbit MD-2;(2) human TLR4 and human MD-2; (3) rabbit TLR4 and human MD-2; (4) humanTLR4 and rabbit MD-2. FIG. 12 shows FACS analysis of these cellsfollowing antibody staining, which revealed that mu16G7 recognized cellsexpressing the human TLR4/MD-2 complex and a combination of human TLR4and rabbit MD-2, but not the rabbit TLR4/MD-2 complex nor a combinationof rabbit TLR4 and human MD-2. These results indicate that the epitoperecognized by mu16G7 is situated on human TLR4 (FIG. 12).

Example 14 mu16G7 VH and VL Sequences

VH and VL sequences from the mu16G7 hybridoma clone were amplified fromtotal RNA by RT-PCR. Sequence analysis is shown in FIGS. 13A-13F.Alignment of the mu16G7 VH and VL nucleotide sequences with known mouseVH and VL sequences (using the International Immunogenetics InformationSystem; which can be found at http://imgt.cines.fr) reveals that themu16G7 VH sequence most closely resembles the IgHV1 subfamily, while themu16G7 VL belongs to the IgKV1 subfamily.

The mu16G7 antibody includes a heavy chain variable region (SEQ IDNO:12, FIG. 13B) encoded by the nucleic acid sequence of SEQ ID NO:11shown in FIG. 13A, and a light chain variable region (SEQ ID NO:17, FIG.13E) encoded by the nucleic acid sequence of SEQ ID NO:16 shown in FIG.13D. The amino acids encompassing the CDR as defined by Chothia et al.1989, E. A. Kabat et al., 1991 are highlighted in underlined anditalicized text in FIGS. 13B and 13E and shown in FIGS. 13C and 13F. Theheavy chain CDRs of the mu16G7 antibody have the following sequences:DYWIE (SEQ ID NO:13); EILPGSGSTNYNEDFKD (SEQ ID NO:14); and EERAYYFGY(SEQ ID NO:15). The light chain CDRs of the mu16G7 antibody have thefollowing sequences: RSSQSLENSNGNTYLN (SEQ ID NO:18); RVSNRFS (SEQ IDNO:19); and LQVTHVPPT (SEQ ID NO:20).

Example 15 Materials and Methods for the Generation of mu15C1 MonoclonalAntibody

A. Generation of Stable TLR4/MD-2 Transfectants

Stable TLR4/MD-2 transfectants were generated in CHO-K1 and HEK 293cells as described above in Example 9, subsection A.

B. Generation of Recombinant MD-2 and Chimeric TLR4/MD-2 Protein

To generate recombinant soluble MD-2, cDNA encoding the protein with Cterminal FLAG and 6×HIS tags for detection and purification purposes wascloned into pFASTBAC1 and subsequently inserted into bacmid DNA byhomologous recombination. Following generation of a viral stock, Sf9cells were superinfected. 48 hours later, the recombinant protein waspurified from infected cell supernatants using a NiNTA affinity matrix(Qiagen).

To generate the recombinant TLR4/MD-2 chimeric protein, cDNA encodingthe extracellular portion of human TLR4 linked to MD-2 via a glycineserine (GGGGS₃) linker was assembled using PCR. FLAG and 6×HIS tags wereincluded at the C-terminus of MD-2 for detection and purificationpurposes. The cDNA cassette was cloned into the baculovirus expressionvector pFASTBAC I (Invitrogen) and subsequently inserted into bacmid DNAby homologous recombination. Following generation of a viral stock, Sf9cells were superinfected. 48 hours later, the recombinant fusion proteinwas purified from cell lysates using an anti-FLAG™ M2 MAb affinitymatrix (Sigma).

C. Immunization of Mice

8 week old female BALB/c mice (IFFA CREDO) were immunized as describedabove in Example 1, subsection C.

D. Specific Serum Titrations

Mice serum titrations were performed as described above in Example 1,subsection D.

E. B Cell/Myeloma Fusions

B cell extraction and cellular fusion were performed and analyzed asdescribed above in Example 9, subsection D.

F. Hybridoma Screening

Hybridoma screening was performed as described above in Example 1,subsection F.

G. Monoclonal Antibody Specificity

The specificity of the mu15C1 monoclonal antibody was determined asdescribed above in Example 1, subsection G.

H. Cellular Assay 1

Cellular Assay I was performed as described above in Example 1,subsection J.

I. Cellular Assay 2

Cellular Assay II was performed as described above in Example 1,subsection K.

J. 15C1 VH and VL Sequences

10⁷ hybridoma cells were harvested and washed once with PBS before beingresuspended in 1 ml Trizol™ reagent (Invitrogen). Total RNA wassubsequently extracted according to the manufacturer's guidelines. cDNAencoding the VH and VL from the mu15C1 clone was generated by RT-PCRwith the mouse ScFv module (Amersham Biosciences) according to themanufacturer's guidelines. Amplified products were cloned into thepGEM-T easy vector (Promega Corp.) and sequenced using the T7 and SP6primers.

The VH and VL cDNAs were subsequently cloned in mammalian expressionvectors containing the human IgG1 and human kappa constant regionsrespectively in order to express mu15C1 as a chimeric MAb (“chimeric15C1”). To produce recombinant chimeric MAb, HEK 293 cells were platedin 6 well plates at a density of 2.5×10⁵ cells/well in 2 ml culturemedium containing 10% FBS. 16 hours post-plating, cells were transfectedwith 0.75 μg of the appropriate vector(s) using Fugene™ reagent (Roche)according to the manufacturer's guidelines. 48 hours post-transfection,supernatant was harvested and antibody was purified using protein Gaffinity chromatography.

Example 16 Generation of MAbs Directed Against the Human TLR4/MD-2Complex

Mice immunized with CHO cells expressing surface TLR4/MD-2 weremonitored for specific serum titers. Those showing a response toTLR4/MD-2 were “hyperboosted” with HEK 293 TLR4/MD-2 transfectants. Thisstrategy was chosen in order to minimize the response to non-specificCHO cellular antigens, while simultaneously maximizing theTLR4/MD-2-specific response. Screening by FACS of supernatants fromhybridomas resulting from B cell/myeloma fusions was performed onmock-transfected vs. TLR4/MD-2-transfected CHO cells. Monoclonalantibody from a specific clone, referred to herein as mu15C1,demonstrated specific binding to TLR4/MD-2 transfected CHO cells (FIG.14). mu15C1 was found to have the IgG1 κ isotype, as determined by FACSusing the mouse Ig isotyping CBA kit (Beckton Dickenson).

Example 17 Neutralization of LPS Activity on TLR4/MD-2 Transfected HEK293 Cells

LPS is known to have ability to induce IL-8 production in HEK 293 cellstransfected with the TLR4/MD-2 complex. The ability of mu15C1 to inhibitthis IL-8 induction was analyzed by pre-incubating cells with eachantibody for 30 minutes prior to LPS administration. FIG. 15 shows thatmu15C1 inhibited the effects of LPS on HEK 293 cells, even atsub-microgram/ml concentrations.

Example 18 Neutralization of LPS Activity on Human Whole Blood

The ability of mu15C1 to inhibit LPS-induced IL-8 production in humanwhole blood was tested. mu15C1 neutralizing activity was tested in bloodfrom 3 different donors using a range of monoclonal antibodyconcentrations from 0.5 to 5 μg /ml. FIG. 16 demonstrates that mu15C1significantly reduced the level of IL-8 induced by LPS in all 3 donors,as compared to an isotype matched control. mu15C1 was found to be morepotent than a previously described α-TLR4 blocking monoclonal antibody(from e-biosciences). (See Shimazu et al. J. Exp. Med. 189: 1777-1782(1999)). In some cases, mu15C1 was found to be as potent as an α-CD14blocking monoclonal antibody that was also included in the study. (SeeKirkland et al. J. Biol. Chem. 268: 24818-24823(1993)). These resultsindicate that the neutralizing epitope recognized by mu15C1 ontransfected HEK 293 cells is also exposed on the surface on cells inwhole blood, and that mu15C1 is potent enough to inhibit the activity ofLPS in whole blood, even at concentrations below 1 μg/ml.

Example 19 mu15C1 Specificity

In order to determine the specificity of the mu15C1 monoclonal antibody,the fact that mu15C1 does not recognize the rabbit ortholog of theTLR4/MD-2 complex (previously cloned) was exploited. cDNAs for eitherrabbit or human TLR4 with N-terminal FLAG™ epitope tag and MD-2 withC-terminal c-Myc and protein C epitope tags were transfected in HEK 293cells in the following combinations: (1) mock vector (2) human TLR4alone (3) human TLR4 and human MD-2 (4) rabbit TLR4 and rabbit MD-2; (5)human TLR4 and rabbit MD-2; (6) rabbit TLR4 and human MD-2. FIG. 17shows FACS analysis of these cells following antibody staining, whichrevealed that mu15C1 recognized cells expressing human TLR4 alone, thehuman TLR4/MD-2 complex and a combination of human TLR4 and rabbit MD-2,but not the rabbit TLR4/MD-2 complex nor a combination of rabbit TLR4and human MD-2. These results indicate that the epitope recognized bymu15C1 is situated on human TLR4 (FIG. 17).

Example 20 mu15C1 VH and VL Sequences

VH and VL sequences from the mu15C1 hybridoma clone were amplified fromtotal RNA by RT-PCR using oligonucleotide primers specific for mouseleader sequences and constant domains (Jones and Bendig, Biotechnology,9: 88-89 (1991)). Sequence analysis is shown in FIGS. 18A-18F.

The mu15C1 antibody includes a heavy chain variable region (SEQ IDNO:22, FIG. 18B) encoded by the nucleic acid sequence of SEQ ID NO:21shown in FIG. 18A, and a light chain variable region (SEQ ID NO:27, FIG.18E) encoded by the nucleic acid sequence of SEQ ID NO:26 shown in FIG.18D. The amino acids encompassing the CDR as defined by Chothia et al.1989, E. A. Kabat et al., 1991 are highlighted in underlined anditalicized text in FIGS. 18B and 18E and shown in FIGS. 18C and 18F. Theheavy chain CDRs of the mu15C1 antibody have the following sequences:GGYSWH (SEQ ID NO:23); YIHYSGYTDFNPSLKT (SEQ ID NO:24); and KDPSDGFPY(SEQ ID NO:25). The light chain CDRs of the mu15C1 antibody have thefollowing sequences: RASQSISDHLH (SEQ ID NO:28); YASHAIS (SEQ ID NO:29);and QNGHSFPLT (SEQ ID NO:30).

Example 21 Chimeric 15C1 Binds to hTLR4 hMD2 Transfected CHO Cells

In order to demonstrate the specificity of the cloned 15C1 VH and VL forthe hTLR4/MD-2 complex, FACS analysis on hTLR4/MD-2 transfected CHOcells using the chimeric 15C1 MAb was performed (FIG. 19). Specificbinding of MAb at the indicated concentration was detected using anAPC-labeled goat-anti-human IgG (H+L) secondary antibody. An irrelevantisotype-matched human IgG1 MAb was used as a control.

Example 22 Chimeric 15C1 Inhibits LPS-Induced IL-8 Production in hTLR4hMD2 Transfected HEK 293 Cells

In order to demonstrate the neutralizing capacity of the cloned 15C1 VHand VL for LPS, the ability of 15C1 to inhibit LPS dependent IL-8induction of hTLR4/MD-2 transfected HEK 293 cells was tested (asdescribed above). FIG. 20 shows that chimeric 15C1 inhibited the effectsof LPS on HEK 293 cells in a manner very similar to that of the 15C1MAb.

Example 23 Materials and Methods for the Generation of mu7E3 MonoclonalAntibody

A. Generation of Stable TLR4/MD-2 Transfectants

Stable TLR4/MD-2 transfectants were generated in CHO-K1 and HEK 293cells as described above in Example 9, subsection A.

B. Generation of Recombinant MD-2 and Chimeric TLR4/MD-2 Protein

Recombinant soluble MD-2 was generated as described above in Example 15,subsection B.

To generate the recombinant TLR4/MD-2 chimeric protein, cDNA encodingthe extracellular portion of human TLR4 linked to MD-2 via a glycineserine (GGGGS₃) linker was assembled using PCR. FLAG and 6×HIS tags wereincluded at the C-terminus of MD-2 for detection and purificationpurposes. The cDNA cassette was cloned into the baculovirus expressionvector pFASTBACI (Invitrogen) and subsequently inserted into bacmid DNAby homologous recombination. Following generation of a viral stock, Sf9cells were superinfected. 48 hours later, the recombinant fusion proteinwas purified from cell lysates using an anti-FLAG™ M2 MAb affinitymatrix (Sigma).

C. Immunization of Mice

8 week old female BALB/c mice (IFFA CREDO) were immunized as describedabove in Example 1, subsection C.

D. Specific Serum Titrations

Mice serum titrations were performed as described above in Example 1,subsection D.

E. B Cell/Myeloma Fusions

B cell extraction and cellular fusion were performed and analyzed asdescribed above in Example 9, subsection D.

F. Hybridoma Screening

Hybridoma screening was performed as described above in Example 1,subsection F.

G. Monoclonal Antibody Specificity

The specificity of the mu7E3 monoclonal antibody was determined asdescribed above in Example 1, subsection G.

H. Cellular Assay 1

Monoclonal antibody was first purified from hybridoma cell supernatantusing protein G affinity chromatography.

Cellular Assay I was performed as described above in Example 1,subsection J.

I. Cellular Assay 2

Cellular Assay II was performed as described above in Example 1,subsection K.

J. 7E3 VH and VL Sequences

10⁷ hybridoma cells were harvested and washed once with PBS before beingresuspended in 1 ml TriZol™ reagent (Invitrogen). Total RNA wassubsequently extracted according to the manufacturer's guidelines. cDNAencoding the VH and VL from the mu7E3 clone was generated by RT-PCRusing oligonucleotide primers specific for mouse leader sequences andconstant domains (Jones and Bendig, Biotechnology, 9: 88-89 (1991)).Amplified products were cloned into the pGEM-T easy vector (PromegaCorp.) and sequenced using the T7 and SP6 primers.

The VH and VL cDNAs were subsequently cloned in mammalian expressionvectors containing the human IgG1 and human kappa constant regionsrespectively in order to express mu7E3 as a chimeric MAb (“chimeric7E3”). To produce recombinant chimeric MAb, HEK 293 cells were plated in6 well plates at a density of 2.5×10⁵ cells/well in 2 ml culture mediumcontaining 10% FBS. 16 hours post-plating, cells were transfected with0.75 μg of the appropriate vector(s) using Fugene™ reagent (Roche)according to the manufacturer's guidelines. 48 hours post-transfection,supernatant was harvested and antibody was purified using protein Gaffinity chromatography.

Example 24 Generation of MAbs Directed Against the Human TLR4/MD-2Complex

Mice immunized with CHO cells expressing surface TLR4/MD-2 weremonitored for specific serum titers. Those showing a response toTLR4/MD-2 were “hyperboosted” with HEK 293 TLR4/MD-2 transfectants. Thisstrategy was chosen in order to minimize the response to non-specificCHO cellular antigens, while simultaneously maximizing theTLR4/MD-2-specific response. Screening by FACS of supernatants fromhybridomas resulting from B cell/myeloma fusions was performed on mocktransfected vs. TLR4/MD-2 transfected CHO cells. Monoclonal antibodyfrom a specific clone, referred to herein as mu7E3, demonstratedspecific binding to TLR4/MD-2 transfected CHO cells (FIG. 21). mu7E3 wasfound to have the IgG1 κ isotype, as determined by FACS using the mouseIg isotyping CBA kit (Beckton Dickenson).

Example 25 Neutralization of LPS Activity on TLR4/MD-2 Transfected HEK293 Cells

LPS is known to have ability to induce IL-8 production in HEK 293 cellstransfected with the TLR4/MD-2 complex. The ability of mu7E3 to inhibitthis IL-8 induction was analyzed by pre-incubating cells with eachantibody for 30 minutes prior to LPS administration. FIG. 22 shows thatmu7E3 inhibited the effects of LPS on HEK 293 cells, even atsub-microgram/ml concentrations.

Example 26 Neutralization of LPS Activity on Human Whole Blood

The ability of mu7E3 to inhibit LPS-induced IL-8 production in humanwhole blood was tested. mu7E3 neutralizing activity was tested in bloodfrom 3 different donors using a range of monoclonal antibodyconcentrations from 0.5 to 5 μg/ml. FIG. 23 demonstrates that mu7E3significantly reduced the level of IL-8 induced by LPS in all 3 donors,as compared to an isotype matched control. mu7E3 was found to be morepotent than a previously described α-TLR4 blocking monoclonal antibody(purchased from e-biosciences). (See Shimazu et al. J. Exp. Med. 189:1777-1782 (1999)). In some cases, mu7E3 was found to be as potent as anα-CD14 blocking monoclonal antibody that was also included in the study.(See Kirkland et al. J. Biol. Chem. 268: 24818-24823(1993)). Theseresults indicate that the neutralizing epitope recognized by mu7E3 ontransfected HEK 293 cells is also exposed on the surface on cells inwhole blood, and that mu7E3 is potent enough to inhibit the activity ofLPS in whole blood, even at concentrations below 1 μg/ml.

Example 27 mu7E3 Specificity

In order to determine the specificity of the mu7E3 monoclonal antibody,the fact that mu7E3 does not recognize the rabbit ortholog of theTLR4/MD-2 complex (previously cloned) was exploited. cDNAs for eitherrabbit or human TLR4 with N-terminal FLAG™ epitope tag and MD-2 withC-terminal c-Myc and protein C epitope tags were transfected in HEK 293cells in the following combinations: (1) mock vector (2) human TLR4alone (3) human TLR4 and human MD-2 (4) rabbit TLR4 and rabbit MD-2; (5)human TLR4 and rabbit MD-2; (6) rabbit TLR4 and human MD-2. FIG. 24shows FACS analysis of these cells following antibody staining, whichrevealed that mu7E3 recognized cells expressing the human TLR4/MD-2complex and a combination of human TLR4 and rabbit MD-2, but not therabbit TLR4/MD-2 complex nor a combination of rabbit TLR4 and humanMD-2. These results indicate that the epitope recognized by mu7E3 issituated human TLR4 but the presence of MD-2 is essential for MAbbinding (FIG. 24).

Example 28 mu7E3 VH and VL Sequences

VH and VL sequences from the mu7E3 hybridoma clone were amplified fromtotal RNA by RT-PCR. Sequence analysis is shown in FIGS. 25A-25F.

The mu7E3 antibody includes a heavy chain variable region (SEQ ID NO:32,FIG. 25B) encoded by the nucleic acid sequence of SEQ ID NO:31 shown inFIG. 25A, and a light chain variable region (SEQ ID NO:37, FIG. 25E)encoded by the nucleic acid sequence of SEQ ID NO:36 shown in FIG. 25D.The amino acids encompassing the CDR as defined by Chothia et al. 1989,E. A. Kabat et al., 1991 are highlighted in underlined and italicizedtext in FIGS. 25B and 25E and shown in FIGS. 25C and 25F. The heavychain CDRs of the mu7E3 antibody have the following sequences: TYNIGVG(SEQ ID NO:33); HIWWNDNIYYNTVLKS (SEQ ID NO:34); and MAEGRYDAMDY (SEQ IDNO:35). The light chain CDRs of the mu7E3 antibody have the followingsequences: RASQDITNYLN (SEQ ID NO:38); YTSKLHS (SEQ ID NO:39); andQQGNTFPWT (SEQ ID NO:40).

Example 29 Chimeric 7E3 Binds to hTLR4 hMD2 Transfected CHO Cells

In order to demonstrate the specificity of the cloned 7E3 VH and VL forthe hTLR4/MD-2 complex, FACS analysis on hTLR4/MD-2 transfected CHOcells using the chimeric 7E3 MAb was performed (FIG. 26). Specificbinding of MAb at the indicated concentration was detected using anAPC-labeled goat-anti-human IgG (H+L) secondary antibody. An irrelevantisotype-matched human IgG1 MAb was used as a control.

Example 30 Chimeric 7E3 Inhibits LPS-Induced IL-8 Production in hTLR4hMD2 Transfected HEK 293 Cells

In order to demonstrate the neutralizing capacity of the cloned 7E3 VHand VL for LPS, the ability of 7E3 to inhibit LPS dependent IL-8induction of hTLR4/MD-2 transfected HEK 293 cells was tested asdescribed above. FIG. 27 shows that chimeric 7E3 inhibited the effectsof LPS on HEK 293 cells.

Example 31 Construction of TLR4/MD-2 Fusion Protein cDNA and Cloninginto pFASTBAC1

The extracellular portion of TLR4 linked to MD-2 via a glycine serine(GGGGS₃) linker was assembled using PCR. FLAG and 6×HIS tags wereincluded at the C-terminus of MD-2 for detection and purificationpurposes. (FIG. 28).

FIGS. 28A-C illustrate the construction of this TLR4/MD-2 fusion proteincDNA according to the present invention. cDNA encoding the extracellularportion of human TLR4 (sTLR4) was amplified by PCR, and unique NheI/XhoIrestriction sites were introduced into 5′ non-annealing primerextensions. The (GGGGS)₃ coding sequence and unique XhoI site wasintroduced into the 5′ non-annealing extension of the sense primer, anda unique HindIII site was introduced into the 5′ non-annealing extensionof the antisense primer. (Panel A). Panel B depicts the sequentialcloning of the amplified sTLR4 and (GGGGS)₃/MD-2 cDNAs into pFASTBAC1between the unique XbaI and HindIII restriction site. Panel C depicts aproposed protein product following expression of the sTLR4/MD-2 cDNA inSf9 cells.

Example 32 Expression of the TLR4/MD-2 Chimeric Protein in SF9 CellLysates and Supernatants

The cDNA cassette of Example 1 was cloned into the baculovirusexpression vector pFASTBAC1 (Invitrogen) and subsequently inserted intobacmid DNA by homologous recombination. Following generation of a viralstock, Sf9 cells were superinfected and expression of the TLR4/MD-2fusion protein was analyzed in the cell lysate at 48 and 72 hours postinfection by Western blotting. (FIG. 29).

FIG. 29 demonstrates the expression of a TLR4/MD-2 chimeric protein ofthe invention in Sf9 cell lysates and supernatants. Protein expressionin the Sf9 cell lysates and supernatants was detected by Westernblotting using the anti-FLAG M2 antibody: Lane 1 depicts cleared lysateat 48 hours post infection; lane 2 depicts cleared lysate at 72 hourspost infection; lane 3 depicts cleared supernatant at 48 hours postinfection; lane 4 depicts cleared supernatant at 72 hours postinfection; and lane 5 contains a reference protein (FLAG tagged). Themolecular weight marker sizes in FIG. 29 are shown in KDa. The predictedmolecular weight of TLR4/MD-2 chimeric protein is approximately 90 KDa,and the appearance of probable degradation product occurs atapproximately 28 KDa.

Example 33 Purification of the TLR4/MD-2 Chimeric Protein from InfectedSF9 Cell Lysates

To purify the fusion protein, Sf9 cells were harvested 48 hours postsuperinfection and lysed in 20 mM Tris pH7.4, 150 mM NaCl, 1% NP40 withCOMPLETE™ protease inhibitors (Roche) at a concentration of 5volumes/gram cells. Following a fifteen hour (15′) incubation at 4° C.,lysates were cleared by centrifugation (4000 rpm) and filtration (0.22μm) and passed through an anti-FLAG M2 MAb affinity matrix (Sigma).Unbound protein was removed from the matrix by successive washing with20 mM Tris (pH 7.4), 150 mM NaCl, 1% NP40 and 20 mM Tris (pH 7.4), 150mM NaCl. Bound protein was eluted from the column with 100 mM glycine(pH 2.75) and collected in 0.5 ml fractions. Fractions were rapidlybrought to neutral pH through the addition of 50 μl of 1M Tris (pH 9).Protein content was analyzed by western blotting (with peroxidaseconjugated anti-FLAG M2) and Coomassie brilliant blue staining. (FIG.30).

FIG. 30 demonstrates the presence of purified TLR4/MD-2 chimeric proteinin infected Sf9 cell lysates. Protein in the cell lysates was detectedby Coomassie brilliant blue staining (FIG. 30, left panel) or Westernblotting (FIG. 30, right panel) using the anti-FLAG M2 antibody. Lanes1-5 depict 0.5 ml eluted fractions from the anti-FLAG M2 affinitycolumn.

Example 34 Inhibition of LPS Induced IL-8 Production using ChimericSoluble TLR4/MD-2

Lipopolysaccharide (LPS) (15 ng/ml) was preincubated with a purifiedchimeric TLR4/MD-2 according to the present invention at varyingconcentrations and subsequently incubated with TLR4/MD-2 transfected HEK293 cells. FIG. 31 is a graph depicting IL-8 production in the cellculture medium 24 hours post treatment.

As seen in FIG. 31, purified chimeric TLR4/MD-2 was shown to have aninhibitory effect on the LPS-induced IL-8 production in TLR4/MD-2transfected HEK cells, thereby indicating that the purified TLR4/MD-2protein of the invention was at least partially conformationallycorrect.

Example 35 Humanization of 18H10, 15C1, and 7E3 Antibodies

Design and Construction of the CDR-Grafted Variable Regions

Mu15C1, mu18H10 and mu7 E3 antibodies were humanized by CDR-grafting(Jones et al, Nature 321 :522-525, 1986 ; Verhofyen et al. Science, 239:1634-1536, 1988). “CDR-grafting” involves redesigning the variableregion so that the amino acids comprising the non-human (i.e., mouse)binding site are integrated into the framework of a human antibodyvariable region. In order to accomplish the humanization process, thechoice of the human framework and the extent of mouse variable regionsequence to be transferred are determined.

The human framework for the humanization process was selected from allpublished sequences for human germline immunoglobulin genes which areused to create the human antibody repertoire (see The internationalImMunoGeneTics database, IMGT, available online). For mu15C1, twocandidates for each V gene were chosen, namely IGHV3-66 (also known asDP-86) and IGHV4-28 (also known as DP-48) for the heavy chain andIGKV3-11 (also known as L6) and IGKV6-21 (also known as A26) for theKappa light chain. For mu7E3, two candidates for the heavy chains werechosen, namely IGHV3-66 (or DP-86) and IGHV2-70 (also known as DP-27)and one candidate for the Kappa light chain IGKV1-12 (also known asL19). For mu18H10 one candidate for each V gene was chosen: IGHV1-69(also known as DP-10) for the heavy chain and IGKV3-11 (or L6) for thelight chain.

The extent of the mouse sequences that are to be transferred isdetermined as follows. Firstly, the antigen binding surface ispredominantly located on a series of loops, known as CDRs, three per Vgene, which extend from the β-barrel framework. In all cases, theresidues chosen for transfer corresponded to the broad definition ofCDRs as defined by Kabat (hypervariable regions; Kabat et al, Sequencesof Proteins of Immunological Interest, Fifth edition, U.S. Department ofHealth and Human Services, U.S. Government Printing Office)) and Chothia(structural loops; Chothia et al, Nature, 342:877-883, 1989). Inaddition, residues not identified in the structural loops orhypervariable regions may contribute to antigen binding directly orindirectly by affecting binding site topology, by inducing a stablepacking of the individual variable domains, or by stabilizing theinter-variable domain interaction. Such residues were identified bysequence alignment analysis and noting “idiosyncratic” residues,followed by examination of their structural location and likely effects.

Once the relevant sequence choices have been made the humanized variableregion DNA were generated using any of the following procedures: by genesynthesis using suitable overlapping oligonucleotides (exemplified byKolbinger et al., Protein Eng. 6, 971-980, 1993)), or by usingsimultaneous or sequential site-directed PCR mutagenesis of existing DNAsequences (Kammann et al., Nucleic Acids Res. 17, 5404). For example,PCR primers coding for the new CDRs were hybridized to a DNA templatethat was a fully human or humanized variable region that was designedbased on the same, or a very similar human variable region (exemplifiedby Sato et al., Cancer Res. 53, 851-856 1993). Several minor variants inthe design of the humanized V genes were obtained using the QuikChangesite directed mutagenesis technique originally described by Stratagene.

Following the construction and sequencing of the DNA sequences codingfor the light and heavy chain leader sequences plus humanized variableregions, the leader-variable regions were converted to humanized wholeIgG genes for expression in mammalian cells by sub-cloning into vectorsthat contain a human light or heavy expression cassette.

Humanized Versions of the 15C1 Antibody

The hu15C1 antibodies of the invention include the variable heavy chain(V_(H)) 4-28 shown below in SEQ ID NO:45 or the V_(H) 3-66 shown belowin SEQ ID NO:46. The hu15C1 antibodies of the invention include thevariable light chain (V_(L)) L6 shown below in SEQ ID NO:47 or A26 shownbelow in SEQ ID NO:48. The amino acids encompassing the complementaritydetermining regions (CDR) as defined by Chothia et al. 1989, E. A. Kabatet al., 1991 are boxed in the sequences provided below. (See Chothia, C,et al., Nature 342:877-883 (1989); Kabat, E A, et al., Sequences ofProtein of immunological interest, Fifth Edition, US Department ofHealth and Human Services, US Government Printing Office (1991)).

Tables 1 and 2 present alignments of the amino acid sequences that wereused to design humanized 15C1 V_(H) regions: TABLE 1 15C1 humanizedHeavy chain 4-28 Kabat Chothia IGMT Mouse Germline Reshaped Number-Number- Number- 15C1 IGHV-4- Version 1 ing ing ing VH 28 15C1 VHComments FR1-1 1 FR1-1 D Q

Vernier zone 2 2 2 V V

LOOP H1 2/11A H2 V 3 3 3 Q Q

4 4 4 L L

5 5 5 Q Q

6 6 6 E E

7 7 7 S S

8 8 8 G G

9 9 9 P P

10 10 11 D G

11 11 12 L L

12 12 13 I V

13 13 14 Q K

14 14 15 P P

15 15 16 S S

16 16 17 Q D

17 17 18 S T

18 18 19 L L

19 19 20 S S

20 20 21 L L

LOOP H1 2/11A H20 L 21 21 22 T T

22 22 23 C C

LOOP H1 2/11A H22 C 23 23 24 T A

Surface residue 24 24 25 V* V*

canonical H1 2(6) LOOP H1 2/11A H24 V 25 25 FR1-26

S

26

G* G*

canonical H1 2(6) LOOP H1 2/11A H26 G 27

Y* Y*

canonical H1 2(6) Vernier zone 28

S S

Vernier zone 29

I* I*

canonical H1 2(6) LOOP H1 2/11A H29 I Vernier zone FR1-30

T S

Vernier zone

G S

G S

LOOP H1 2/11A H31A D but G in 15C1

Y N

S W

LOOP H1 2/11A H33 A but S in 15C1

34 39 W* W*

canonical H1 2(6) LOOP H1 2/11A H34 W VH/VL interface

35 40

G

FR2-36 36 41 W W

LOOP H1 2/11A H36 W 37 37 42 I I

VH/VL interface 38 38 43 R R

39 39 44 Q Q

VH/VL interface 40 40 45 F P

41 41 46 P P

42 42 47 G G

43 43 48 N K

44 44 49 K G

45 45 50 L L

VH/VL interface (+) 46 46 51 E E

47 47 52 W W

LOOP H2 1/9A H47 WY VH/VL interface Vernier zone 48 48 53 M I

LOOP H1 2/11A H48 M Vernier zone FR2-49 49 54 G G

Vernier zone

50 55 Y Y

LOOP H1 2/11A H50 Y Vernier zone

51

I I

LOOP H2 1/9A H51 IMV

H Y

Y Y

LOOP H1 2/11A H53 Y

S S

G* G*

canonical H2 class 1 (16) GD LOOP H2 1/9A H55 G

Y S

57

T T

58 66 D Y

59 67 F Y

LOOP H2 1/9A H59 YL But F in 15C1

60 68 N N

61 69 P P

62 70 S S

63 71 L L

64 72 K K

65 74 T S

FR3-66 66 75 R R

67 67 76 I V

Vernier zone Clos to CDRs 68 68 77 S T

69 69 78 I M

LOOP H1 2/11A H69 I LOOP H2 1/9A H69 IM Vernier zone 70 70 79 T S

71 71 80 R*

canonical H2 class 1(16) RKVI LOOP H2 1/9A H71 Vernier zone 72 72 811 DD

73 73 82 T T

Vernier zone 74 74 83 S S

75 75 84 K K

76 76 85 N N

LOOP H1 2/11A H76 N 77 77 86 Q Q

78 78 87 F F

LOOP H1 2/11A H78 F Vernier zone 79 89 88 F S

80 80 89 L L

LOOP H1 2/11A H80 L 81 81 90 Q K

82 82 91 L L

82A 82A 92 N S

82B 82B 93 S S

82C 82C 94 V V

83 83 95 T T

84 84 96 T A

85 85 97 E V

86 86 98 D D

87 87 99 T T

88 88 100 A A

89 89 101 T V

90 90 102 Y Y

91 91 103 Y Y

VH/VL interface 92 92 104 C C

LOOP H1 2/11A H92 C 93 93

A A

VH/VL interface Vernier zone FR3-94 94

R* R*

canonical H1 2(6) Vernier zone

VH/VL interface

LOOP H1 2/11A H96 W But D in 15C1

VH/VL interface (+)

FR4-103 103 W

VH/VL interface (+) Vernier zone 104 104 G

105 105 Q

106 106 G

107 107 T

108 108 L

109 109 V

110 110 T

111 111 V

112 112 S

FR4-113 113 A

Legend: The first column (Kabat numbering) gives the residue numberaccording to Kabat et al. (1991); the second column (Chothia numbering)gives the residue number according to Chothia; the third column (IMGTnumbering) gives the IMGT unique Lefranc numbering for 15C1 V_(H); thefourth column (mouse 15C1 V_(H)) gives the amino acid sequence of theV_(H) region of mouse 15C1 anti-TLR4 MD2 antibody used as donor sequencefor CDR-grafting; the fifth column (Human Germline IGHV4-28) gives thesequence amino acid of the human germline immunoglobulin heavy variable4-28 used as acceptor sequence for CDR-grafting; and the sixth column(Reshaped version 1 15C1 VH) gives the amino acid sequence of humanizedversion of 15C1 V_(H) region. The positions of framework segments (FR1,FR2, FR3, and FR4) and the complementarity- determining segments (CDR1,CDR2, and CRD3) with are shown in column one.

As used in Tables 1, (*) indicates parts of main canonical structure forthe CDR loops defined by Chothia et al. (1989). The bolded entries withno underlining represent positions in FRs and CDRs where the human andmouse amino acid residues are identical. The italicized entriesrepresent positions in FRs where the human residue differs from theanalogous mouse residue number. The underlined entries (bolded or notbolded) represent positions in FRs and CDRs where the human amino acidresidue was replaced by the corresponding mouse residue. The boxedentries represent human residues conserved in the humanized version.TABLE 2 15C1 humanized Heavy chain 3-66 Human- Kabat Chothia IGMT MouseGermline ized Number- Number- Number- 15C1 IGHV3- version ing ing ing VH66 3-66 Commants FR1-1 1 FR1-1 D E

Vernier zone 2 2 2 V V

LOOP H1 2/11A H2 V 3 3 3 Q Q

4 4 4 L L

5 5 5 Q V

6 6 6 E E

7 7 7 S S

8 8 8 G G

9 9 9 P G

10 10 11 D G

11 11 12 L L

12 12 13 I V

13 13 14 Q Q

14 14 15 P P

15 15 16 S G

16 16 17 Q G

17 17 18 S S

18 18 19 L L

19 19 20 S R

20 20 21 L L

LOOP H1 2/11A H20 L 21 21 22 T S

22 22 23 C C

LOOP H1 2/11A H22 C 23 23 24 T A

Surface residue 24 24 25 V* A

canonical H1 2(6) LOOP H1 2/11A H24 V 25 25 FR1-26 T S

26

G* G

canonical H1 2(6) LOOP H1 2/11A H26 G 27

F

canonical H1 2(6) Vernier zone 28

S T

Vernier zone 29

V

canonical H1 2(6) LOOP H1 2/11A H29 I Vernier zone FR1-30

T S

Vernier zone

G S

G N

LOOP H1 2/11A H31 A D but G in 15C1

Y Y

S M

LOOP H1 2/11A H33 A but S in 15C1

34 39 W*

canonical H1 2(6) LOOP H1 2/11A H34 W VH/VL interface

35 40 H S

FR2-36 36 41 W W

LOOP H1 2/11A H36 W 37 37 42 I V

VH/VL interface 38 38 43 R R

39 39 44 Q Q

VH/VL interface 40 40 45 F A

41 41 46 P P

42 42 47 G G

43 43 48 N K

44 44 49 H G

45 45 50 L L

VH/VL interface (+) 46 46 51 E E

47 47 52 W W

LOOP H2 1/9A H47 WY VH/VL interface Vernier zone 48 48 53 M V

LOOP H1 2/11A H49 M Vernier zone FR2-49 49 54 G S

Vernier zone

50 55 Y V

LOOP H1 2/11A H50 Y Vernier zone

51

I I

LOOP H2 1/9A H51 IMV

H Y

Y S

LOOP H1 2/11A H53 Y

S G

G* G

canonical H2 class 1 (16) GD LOOP H2 1/9A H55 G

Y S

57

T T

58 66 D Y

59 67 F Y

LOOP H2 1/9A H59 YL But F in 15C1

60 68 N A

61 69 P D

62 70 S S

63 71 L V

64 72 K K

65 74 T G

FR3-66 66 75 R R

67 67 76 I F

Vernier zone Close to CDRs 68 68 77 S T

69 69 78 I I

LOOP H1 2/11A H69 I LOOP H2 1/9A H69 IM Vernier zone 70 70 79 T S

71 71 80 R* R

canonical H2 class 1(16) RKVI LOOP H2 1/9A H71 RKV Vernier zone 72 72 81D D

73 73 82 T N

Vernier zone 74 74 83 S S

75 75 84 K K

76 76 85 N N

LOOP H1 2/11A H76 N 77 77 86 Q T

78 78 87 F L

LOOP H1 2/11A H78 F Vernier zone 79 89 88 F Y

80 80 89 L L

LOOP H1 2/11A H80 L 81 81 90 Q Q

82 82 91 L M

82A 82A 92 N N

82B 82B 93 S S

82C 82C 94 V L

83 83 95 T R

84 84 96 T A

85 85 97 E E

86 86 98 D D

87 87 99 T T

88 88 100 A A

89 89 101 T V

90 90 102 Y Y

91 91 103 Y Y

VH/VL interface 92 92 104 C C

LOOP H1 2/11A H92 C 93 93

A A

VH/VL interface Vernier zone FR3-94 94

R* R

canonical H1 2(6) Vernier zone

VH/VL interface

LOOP H1 2/11A H96 W But D in 15C1

G Y

F F

VH/VL interface (+)

P D

Y Y

FR4-103 103 W W

VH/VL interface (+) Vernier zone 104 104 G G

105 105 Q Q

106 106 G G

107 107 T T

108 108 L L

109 109 V V

110 110 T T

111 111 V V

112 112 S S

FR4-113 113 A S

Legend: The first column (Kabat numbering) gives the residue numberaccording to Kabat et al (1991); the second column (Chothia numbering)gives the residue number according to Chothia; the third column (IMGTnumbering) gives the IMGT unique Lefranc numbering for 15C1 VH; thefourth column (mouse 15C1 VH) gives the amino acid sequence of the V_(H)region of mouse 15C1 anti-TLR4 MD2 antibody used as donor sequence forCDR-grafting; the fifth column (Human Germline IGHV 3-66) gives thesequence amino acid of the human germline immunoglobulin heavy variable3-66 used as acceptor sequence for CDR-grafting; and the sixth column(Humanized version 3-66) gives the amino acid sequence of the humanizedversion of 15C1 V_(H) region. The positions of framework segments (FR1,FR2, FR3, and FR4) and the complementarity- determining segments (CDR1,CDR2, and CDR3) with are shown in column one.

As used in Table 2, (*) indicates parts of main canonical structure forthe CDR loops as defined by Chothia et al. (1989). The bolded entries,not underlined, represent positions in FRs and CDRs where the human andmouse amino acid residues are identical. The italicized entriesrepresent positions in FRs where the human residue differs from theanalogous mouse residue number. The underlined entries (bolded or notbolded) represent positions in FRs and CDRs where the human amino acidresidue was replaced by the corresponding mouse residue. The boxedentries represent human residues conserved in the humanized version.

Tables 3 and 4 present alignments of the amino acid sequences that wereused to design the humanized 15C1 V_(L) regions: TABLE 3 15C1 humanizedLight chain A26 Human Germline Mouse A26 Humanized FR or 15C1 IGKV6-15C1 VL Kabat # CDR Light 21 A26 Comments  1  1 FR1 D E E  2  2 | I I IL1 class 2/11A (11) I  3  3 | V V V  4  4 | M L L Vernier zone  5  5 | TT T  6  6 | Q Q Q  7  7 | S S S  8  8 | P P P  9  9 | A D D 10 10 | T FF 11 11 | L Q Q 12 12 | S S S 13 13 | V V V 14 14 | T T T 15 15 | P P P16 16 | G K K 17 17 | D E E 18 18 | R K K 19 19 | V V V 20 20 | S T T 2121 | L I I 22 22 | S T T 23 23 FR1 C C C 24 24 CDR1 R R R 25 25 | A A AL1 class 2/11A (11) A 26 26 | S S S 27 27 | Q Q Q 28 28 | S S S 29 29 |I I I L1 class 2/11A (11) IV 30 30 | S G S 31 31 | D S D 32 32 | H S H33 38 | L L L L1 class 2/11A (11) L 34 34 CDR1 H H H 35 35 FR2 W W W L1class 2/11A (11) W 36 36 | Y Y Y VH/VL inter Vernier zone 37 37 | Q Q Q38 38 | Q Q Q VL/VH inter 39 39 | K K K 40 40 | S P P 41 41 | H D D 4242 | E Q Q 43 43 | S S S 44 44 | P P P VL/VH inter+ 45 45 | R K K 46 46| L L L VL/VH inter Vernier zone 47 47 | L L L Vernier zone 48 48 | I II L2 class 1/7A (7) IV 49 49 FR2 K K K Vernier zone 50 50 CDR2 Y Y Y 5151 | A A A 52 52 | S S S 53 53 | H Q H 54 54 | A S A 55 55 | I F I 56 56CDR2 S S S 57 57 FR3 G G G 58 58 | I V V 59 59 | P P P 60 60 | S S S 6161 | R R R 62 62 | F F F 63 63 | S S S 64 64 | G G G L2 class 1/7A (7) G65 65 | S S S 66 66 | G G G Vernier zone 67 67 | S S S 68 68 | G G GVernier zone 69 69 | T T T Vernier zone 70 70 | D D D 71 71 | F F F L1class 2/11A (11) YF 72 72 | T T T 73 73 | L L L 74 74 | S T T 75 75 | II I 76 76 | K N N 77 77 | S S S 78 78 | V L L 79 79 | E E E 80 80 | P AA 81 81 | E E E 82 82 | D D D 83 83 | I A A 84 84 | G A A 85 85 | V T T86 86 | Y Y Y 87 87 | Y Y Y VL/VH inter 88 88 FR3 C C C 89 89 CDR3 Q H QVL/VH inter 90 90 | N Q N L3 class 1/9A (9) QNH 91 91 | G S G VL/VHinter 92 92 | H S H 93 93 | S S S 94 94 | E L F 95 95 | P P P L3 class1/9A (9) P 96 96 | L L L VL/VH inter+ 97 97 CDR3 T T T 98 98 FR4 F F FVL/VH inter+ Vernier zone 99 99 | G G G 100  100  | A G G 101  101  | GG IGKJ4 G IGKJ4 102  102  | T T T 103  103  | K K K 104  104  | L V V105  105  | E E E 106  106  | L I I 107  107  FR4 K K K

Legend: The first column (Kabat) gives the residue number according toKabat et al. (1991); The second column (#) gives the residue number inregular sequence; The third column (FR or CDR) is convenient to identifythe framework segments (FR1, FR2, FR3, and FR4) and thecomplementarity-determining segments (CDR1, CDR2, and CDR3) with thethree CDRs separating the four FRs; The fourth column (mouse 15C1 Light)gives the amino acid sequence of the V_(L) region of mouse 15C1antibody; The fifth column (Human GermalineA26 IGKV6-21) gives thesequence amino acid of the human germline Kappa light chain A26 orIGKV6-21; The sixth column (Humanized 15C1 VL A26) gives the amino acidsequence of humanized version of 15C1 VL A26.

As used in Table 3, (*) represents part of main canonical structure forthe CDR loops as defined by Chothia et al. (1989). Bolded entries, notunderlined, represent positions in FRs and CDRs where the human andmouse amino acid residues are identical. Italicized entries representpositions in FRs where the human residue differs from the analogousmouse residue number. Underlined entries (bolded or not bolded)represent positions in FRs and CDRs where the human amino acid residuewas replaced by the corresponding mouse residue. TABLE 4 15C1 HumanizedLight chain L6 Human Mouse Germline Humanize FR or 15C1 L6 d 15C1 VLKabat # CDR Light IGKV3-11 L6 Comments  1  1 FR1 D E E  2  2 | I I I* L1class 2/11A (11) I  3  3 | V V V  4  4 | M L L Vernier zone  5  5 | T TT  6  6 | Q Q Q  7  7 | S S S  8  8 | P P P  9  9 | A A A 10 10 | T T T11 11 | L L L 12 12 | S S S 13 13 | V L L 14 14 | T S S 15 15 | P P P 1616 | G G G 17 17 | D E E 18 18 | R R R 19 19 | V A A 20 20 | S T T 21 21| L L L 22 22 | S S S 23 23 FR1 C C C 24 24 CDR1 R R R 25 25 | A A A* L1class 2/11A (11) A 26 26 | S S S 27 27 | Q Q Q 28 28 | S S S 29 29 | I VI* L1 class 2/11A (11) IV 30 30 | S S S 31 31 | D S D 32 32 | H Y H 3333 | L L L* L1 class 2/11A (11) L 34 34 CDR1 H A H 35 35 FR2 W W W* L1class 2/11A (11) W 36 36 | Y Y Y VH/VL inter Vernier zone 37 37 | Q Q Q38 38 | Q Q Q VL/VH inter 39 39 | K K K 40 40 | S P P 41 41 | H G G 4242 | E Q Q 43 43 | S A A 44 44 | P P P VL/VH inter+ 45 45 | R R R 46 46| L L L VL/VH inter Vernier zone 47 47 | L L L Vernier zone 48 48 | I II* L2 class 1/7A (7) IV 49 49 FR2 K Y K OR Y Vernier zone 50 50 CDR2 Y DY 51 51 | A A A 52 52 | S S S 53 53 | H N H 54 54 | A R A 55 55 | I A I56 56 CDR2 S T S 57 57 FR3 G G G 58 58 | I I I 59 59 | P P P 60 60 | S AA 61 61 | R R R 62 62 | F F F 63 63 | S S S 64 64 | G G G* L2 class 1/7A(7) G 65 65 | S S S 66 66 | G G G Vernier zone 67 67 | S S S 68 68 | G GG Vernier zone 69 69 | T T T Vernier zone 70 70 | D D D 71 71 | F F F*L1 class 2/11A (11) YF 72 72 | T T T 73 73 | L L L 74 74 | S T T 75 75 |I I I 76 76 | K S S 77 77 | S S S 78 78 | V L L 79 79 | E E E 80 80 | PP P 81 81 | E E E 82 82 | D D D 83 83 | I F F 84 84 | G A A 85 85 | V VV 86 86 | Y Y Y 87 87 | Y Y Y VL/VH inter 88 88 FR3 C C C 89 89 CDR3 Q QQ VL/VH inter 90 90 | N Q N* L3 class 1/9A (9) QNH 91 91 | G R G VL/VHinter 92 92 | H S H 93 93 | S N S 94 94 | F W F 95 95 | P P P* L3 class1/9A (9) P 96 96 | L L L VL/VH inter+ 97 97 CDR3 T T T 98 98 FR4 F F FVL/VH inter+ Vernier zone 99 99 | G G G 100  100  | A G G 101  101  | GG IGKJ4 G 102  102  | T T T 103  103  | K K K 104  104  | L V V 105 105  | E E E 106  106  | L I I 107  107  FR4 K K K

Legend: The first column (Kabat) gives the residue number according toKabat et al. (1991); The second column (#) gives the residue number inregular sequence; The third column (FR or CDR) is convenient to identifythe framework segments (FR1, FR2, FR3, and FR4) and thecomplementarity-determining segments (CDR1, CDR2, and CDR3) with thethree CDRs separating the four FRs; The fourth column (mouse 15C1 Light)gives the amino acid sequence of the V_(L) region of mouse 15C1anti-TLR4 MD2 antibody; The fifth column (Human Germline L6 or IGKV3-11)gives the sequence amino acid of the human germline Kappa light chain L6or IGKV3. The sixth column (Humanized 15C1 VL L6) gives the amino acidsequences of humanized 15C1 light chain.

As used in Table 4, (*) represents part of main canonical structure forthe CDR loops as defined by Chothia et al. (1989). Bolded entries, notunderlined, represent positions in FRs and CDRs where the human andmouse amino acid residues are identical. Italicized entries representpositions in FRs where the human residue differs from the analogousmouse residue number. Underlined entries (bolded or not bolded)represent positions in FRs and CDRs where the human amino acid residuewas replaced by the corresponding mouse residue.

Humanized Versions of the 18H10 Antibody

The hu18H10 antibodies of the invention include the V_(H) 1-69 shownbelow in SEQ ID NO:49. The hu18H10 antibodies of the invention includethe V_(L) L6 shown below in SEQ ID NO:50. The amino acids encompassingthe complementarity determining regions (CDR) as defined by Chothia etal. 1989, E. A. Kabat et al., 1991 are boxed in the sequences providedbelow. (See Chothia, C, et al., Nature 342:877-883 (1989); Kabat, E A,et al., Sequences of Protein of immunological interest, Fifth Edition,US Department of Health and Human Services, US Government PrintingOffice (1991)).

Table 5 presents alignments of the amino acid sequences that were usedto design the humanized 15C1 V_(H) region: TABLE 5 18H10 Humanized Heavychain 1-69 Kabat Chothia IGMT Mouse Human Humanized Number- Number-Number- 18H10 Germline 18H10 ing ing ing VH UGHV1-69 VH 1-69 CommentsFR1-1 1 FR1-1 E Q

Vernier zone 2 2 2 V V

LOOP H1 1/10A H2 VIG 3 3 3 Q Q

4 4 4 L L

LOOP H1 1/10A H4 LV 5 5 5 Q V

6 6 6 Q Q

7 7 7 S S

8 8 8 G G

9 9 9 A A

10 10 11 D E

11 11 12 L V

12 12 13 V K

13 13 14 R K

13 13 14 R K

14 14 15 P P

15 15 16 G G

16 16 17 A S

17 17 18 L S

18 18 19 V V

19 19 20 K K

20 20 21 L V

LOOP H1 1/10A H20 LIMV 21 21 22 S S

22 22 23 C C

LOOP H1 1/10A H22 C 23 23 24 T K

Surface residue 24 24 25 A* A*

canonical H1 2(6) LOOP H1 1/10A H24 TAVGS 25 25 FR1-26 S S

26

G* G*

canonical H1 2(6) LOOP H1 1/10A H26 G 27

F* G*

canonical H1 2(6) Vernier zone 28

N T

Vernier zone 29

I* F*

canonical H1 2(6) LOOP H1 1/10A H29 IFLS Vernier zone FR1-30

K S

Vernier zone

D S

S Y

LOOP H1 1/10A H31 A IHYFTNCED but S in 18H10

33

Y A

LOOP H1 1/10A H33 YAWGTLV LOOP H2 2/10A H33 YWGATL

34

I I

LOOP H1 1/10A IVMW

35 39 H* S*

canonical H1 2(6) LOOP H1 1/10A H35 HENQSYT VH/VL interface FR2-36 36 41W W

LOOP H1 1/10A H36 W 37 37 42 V V

VH/VL interface 38 38 43 K R

39 39 44 K Q

VH/VL interface 40 40 45 R A

41 41 46 P P

42 42 47 E G

43 43 48 W Q

44 44 49 G G

45 45 50 L L

VH/VL interface (+) 46 46 51 E E

47 47 52 W W

LOOP H2 2/10A H47 WY VH/VL interface Vernier zone 48 48 53 I M

LOOP H1 1/10A H48 IMVL Vernier zone FR2-49 49 54 G G

Vernier zone

50 55 W G

LOOP H2 2/10A H50 REWYGQVLNKA Vernier zone

51

T I

LOOP H1 1/10A H51 LIVTSN LOOP H2 2/10A H51 LI but T for 18H10

D I

LOOP H2 2/10A H52 DLNSY

P P P

E I

LOOP H2 2/10A H53 AGYSKTN but E for 18H10

N F

LOOP H2 2/10A H54 NSTKDG

V* G*

N T

LOOP H2 2/10A H56 YREDGVSA but N for 18H10

57

S A

58 66 I N

LOOP H2 2/10A H58 KNTSDRGFY but I for 18H10

59 67 Y Y

LOOP H2 2/10A H59 Y

60 68 D A

61 69 P Q

62 70 R K

63 71 F F

64 72 Q Q

65 74 G G

FR3-66 66 75 K R

67 67 76 A V

Vernier zone Close to CDRs 68 68 77 S T

69 69 78 I I

LOOP H1 1/10A ILFMV Very unusual residue LOOP H2 2/10A H69 IFLM But F in7E3 Vernier zone 70 70 79 T T

71 71 80 A* A*

canonical H2 class 1(16) RKVI LOOP H2 2/10A H71 VAL Vernier zone 72 7281 D D

73 73 82 T K

Vernier zone 74 74 83 S S

75 75 84 S T

76 76 85 N S

77 77 86 T T

78 78 87 A A

LOOP H1 1/1A H78 ALVYF LOOP H2 H78 ALV Vernier zone 79 89 88 F Y

80 80 89 L M

LOOP H1 1/10A H80 LM 81 81 90 Q E

82 82 91 L L

82A 82A 92 T S

82B 82B 93 S S

82C 82C 94 L L

83 83 95 T R

84 84 96 S S

85 85 97 E E

86 86 98 D D

87 87 99 T T

88 88 100 A A

89 89 101 V V

90 90 102 Y Y

LOOP H1 1/10A H90 YF 91 91 103 Y Y

VH/VL interface 92 92 104 C C

LOOP H1 1/10A H92 C 93 93

A A

VH/VL interface Vernier zone FR3-94 94

R* R*

LOOP H1 1/10A J94 RKFSHN canonical H1 2(6) Vernier zone

G

VH/VL interface

Y

N

G

V

Y Y

Y Y

VH/VL interface (+)

A

M M

D D

Y

LOOP H1 1/10A H102 YHVISDG FR4-103 103 W

VH/VL interface (+) Vernier zone 104 104 G

105 105 Q

106 106 G

107 107 T

108 108 T

109 109 V

110 110 T

111 111 V

112 112 S

FR4-113 113 S

Legend: The first column (Kabat numbering) gives the residue numberaccording to Kabat et al. (1991); The second column (Chothia numbering)gives the residue number according to Chothia; The third column (IMGTnumbering) gives the IMGT unique Lefranc numbering for 15C1 VH. Thefourth column (mouse 15C1 VH) gives the amino acid sequence of the V_(H)region of mouse 15C1 anti-TLR4 MD2 antibody used as donor sequence forCDR-grafting; The fifth column (Human Germline IGHV1-69) gives thesequence amino acid of the human germline immunoglobulin heavy variable1-69 (IMGT denomination) used as acceptor sequence for CDR-grafting. Thesixth column (Humanized 18H10 VH 1-69) gives the amino acid sequence ofthe humanized version of 18H10 heavy chain. The positions of frameworksegments (FR1, FR2, FR3, and FR4) and the complementarity- determiningsegments (CDR1, CDR2, and CDR3) with are shown in column one.

As used in Table 5, (*) indicates parts of main canonical structure forthe CDR loops as defined by Chothia et al. (1989). Bolded entries, notunderlined, represent positions in FRs and CDRs where the human andmouse amino acid residues are identical. Italicized entries representpositions in FRs where the human residue differs from the analogousmouse residue number. Underlined entries (bolded or not bolded)represent positions in FRs and CDRs where the human amino acid residuewas replaced by the corresponding mouse residue. Boxed entries representhuman residues conserved in the humanized version.

Table 6 presents alignments of the amino acid sequences that were usedto design the humanized 15C1 V_(L) region: TABLE 6 18H10 humanized Lightchain L6 Human Mouse Germline Humanized FR or 18H10 L6 18H10 Kabat # CDRLight IGKV3-11 VL L6 Comments  1  1 FR1 Q E

 2  2 | I I

L1 class 1/10 I  3  3 | V V

 4  4 | L L

Vernier zone  5  5 | T T

 6  6 | Q Q

 7  7 | S S

 8  8 | P P

 9  9 | S A

 10  10 | I T

 11  11 | M L

 12  12 | S S

 13  13 | A L

 14  14 | S S

 15  15 | L P

 16  16 | G G

 17  17 | E E

 18  18 | E R

 19  19 | I A

 20  20 | T T

 21  21 | L L

 22  22 | T S

 23  23 FR1 C C

L1 class 1/10 C   24   24 CDR1 S R S  25  25 | A A

L1 class 1/10 A  26  26 | S S

 27  27 | S Q S  28  28 | S S

 29  29 | V  30  30 | V S V* L1 class 1/10 V  31  31 | I S I  32  32 | YY

 33  38 | M L M* L1 class 1/10 LM   34   34 CDR1 H A H  35  35 FR2 W W

L1 class 1/10 W  36  36 | Y Y

VH/VL inter Vernier zone  37  37 | Q Q

 38  38 | Q Q

VL/VH inter  39  39 | K K

 40  40 | S P

 41  41 | G G

 42  42 | T Q

 43  43 | S A

 44  44 | P P

VL/VH inter+  45  45 | K R

 46  46 | L L

VL/VH inter Vernier zone  47  47 | L L

Vernier zone  48  48 | I I

L2 class 1/7A (7) IV  49  49 FR2 Y Y

Vernier zone   50   50 CDR2 R D R  51  51 | T A T  52  52 | Y S Y  53 53 | N N

 54  54 | L R L  55  55 | A A

  56   56 CDR2 S T S  57  57 FR3 G G

 58  58 | V I

 59  59 | P P

 60  60 | S A

 61  61 | R R

 62  62 | F F

 63  63 | S S

 64  64 | G G

L2 class 1/7A (7) G  65  65 | S S

 66  66 | G G

Vernier zone  67  67 | S S

 68  68 | G G

Vernier zone  69  69 | T T

Vernier zone  70  70 | F D

 71  71 | Y F

L1 class 1/10 Y  72  72 | S T

 73  73 | L L

 74  74 | T T

 75  75 | I I

 76  76 | S S

 77  77 | S S

 78  78 | V L

 79  79 | E E

 80  80 | A P

 81  81 | E E

 82  82 | D D

 83  83 | A F

 84  84 | A A

 85  85 | D V

 86  86 | Y Y

 87  87 | Y Y

 88  88 FR3 C C

  89   89 CDR3 H Q H VL/VH inter  90  90 | Q Q

L3 class 1/9A (9) QNH  91  91 | W R W VL/VH inter  92  92 | S S

 93  93 | S N S  94  94 | F W F  95  95 | P P

L3 class 1/9A (9) P  96  96 | Y

VL/VH inter+   97   97 CDR3 T

 98  98 FR4 F

VL/VH inter+Vernier zone  99  99 | G

100 100 | G

101 101 | G

102 102 | T

103 103 | K

104 104 | L

105 105 | E

106 106 | I

107 107 FR4 K

Legend: The first column (Kabat) gives the residue number according toKabat et al. (1991); The second column (#) gives the residue number inregular sequence; The third column (FR or CDR) is convenient to identifythe framework segments (FR1, FR2, FR3, and FR4) and thecomplementarity-determining segments (CDR1, CDR2, and CDR3) with thethree CDRs separating the four FRs; The fourth column (mouse 18H10Light) gives the amino acid sequence of the V_(L) region of mouse 18H10anti-TLR4 MD2 antibody; The fifth column (Human Germline L6 or IGKV3-11)gives the sequence amino acid of the human germline Kappa light chain L6or IGKV3. The sixth column (Humanized 18H10 VL L6) gives the amino acidsequences of humanized 18H10 light chain.

As used in Table 6, (*) represents part of main canonical structure forthe CDR loops as defined by Chothia et al. (1989). Bolded entries, notunderlined, represent positions in FRs and CDRs where the human andmouse amino acid residues are identical. Italicized entries representpositions in FRs and CDRs where human residues differ from analogousmouse residues numbers. Underlined entries (bolded or not bolded)represent positions in FRs and CDRs where the human amino acid residuewas replaced by the corresponding mouse residue. Boxed entries representhuman residues conserved in the humanized version.

Humanized Versions of the 7E3 Antibody

The hu7E3 antibodies of the invention include the V_(H) 2-70 shown belowin SEQ ID NO:51 or the V_(H) 3-66 shown below in SEQ ID NO:52. The hu7E3antibodies of the invention include the V_(L) L19 shown below in SEQ IDNO:53. The amino acids encompassing the complementarity determiningregions (CDR) as defined by Chothia et al. 1989, E. A. Kabat et al.,1991 are boxed in the sequences provided below. (See Chothia, C, et al.,Nature 342:877-883 (1989); Kabat, E A, et al., Sequences of Protein ofimmunological interest, Fifth Edition, US Department of Health and HumanServices, US Government Printing Office (1991)).

Tables 7 and 8 present alignments of the amino acid sequences that wereused to design the humanized 15C1 V_(H) regions: TABLE 7 7E3 humanizedHeavy chain 3-66 Version Kabat Chothia IGMT Mouse Germline 3-66Numbering Numbering Numbering 7E3VH IGHV3-66 7E3 VH Comments  FR1-1  1FR1-1 Q E E Vernier zone  2  2  2 V V V  3  3  3 T Q Q  4  4  4 L L L  5 5  5 K V V  6  6  6 E E E  7  7  7 S S S  8  8  8 G G G  9  9  9 P G G10 10 11 G G G 11 11 12 I L L 12 12 13 L V V 13 13 14 Q Q Q 14 14 15 P PP 15 15 16 S G G 16 16 17 Q G G 17 17 18 T S S 18 18 19 L L L 19 29 20 SR R 20 20 21 L L L LOOP H1 3/12A H20 L 21 21 22 T S S 22 22 23 C C CLOOP H1 3/12A H22 C 23 23 24 S A A Surface residue 24 24 25 F* A F* or Acanonical H1 2(6) LOOP H1 3/12A H24 VF 25 25 FR1-26 S S S 26 26 27 G* GG* canonical H1 2(6) CDR1 IMGT LOOP H1 3/12A H26 G Chothia CDR1 27 27 28F* F F* canonical H1 2(6) Vernier zone 28 28 29 S T S LOOP H1 3/12A H28S Vernier zone 29 29 30 L* V L* canonical H1 2(6) LOOP H1 2/11A H29 ILVernier zone FR1-30 30 31 T S T Vernier zone 31 31 32 T S T CDR1 Kabat32 31A 33 Y N Y LOOP H1 2/11A H31A D but G in 15C1 33 31B 34 N Y N 34 3235 I M I CDR1 Chothia 35 33 36 G* S G* canonical H1 2(6) IMGT LOOP H12/11A H34 WV CDR1 VH/VL interface 35A 34 39 V V 35B 35 40 G G CDR1 KabatFR2-36 36 41 W W W LOOP H1 2/11A H36 W 37 37 42 I V V VH/VL interface 3838 43 R R R 39 39 44 Q Q Q VH/VL interface 40 40 45 P A A 41 41 46 S P P42 42 47 G G G 43 43 48 K K K 44 44 49 G G G 45 45 50 L L L VH/VLinterface (+) 46 46 51 E E E 47 47 52 W W W LOOP H2 1/9A H47 WY VH/VLinterface Vernier zone 48 48 53 L V L or V LOOP H1 3/12A H48 ML Vernierzone FR2-49 49 54 A S S Vernier zone 50 50 55 H V H Vernier zone CDR2Kabat 51 51 56 I I I LOOP H2 1/9A H51 IMV CDR2 IMGT 52 52 57 W Y W CDR2Chothia 53 53 58 W S W LOOP H1 3/12A H53 YW 54 54 59 N G N 55 55 60 D* GD* canonical H2 class 1 (16) GD LOOP H2 1/9A H55 G But D in 7E3 56 56 61N S N CDR2 Chothia 57 57 62 I T I CDR2 IMGT 58 58 66 Y Y Y 59 59 67 Y YY LOOP H2 1/9A H59 YL 60 60 68 N A N 61 61 69 T D T 62 62 70 V S V 63 6371 L V L 64 64 72 K K K 65 65 74 S G S CDR2 Kabat FR3- 66 66 75 R R R 6767 76 L F L or F Vernier zone Close to CDRs 68 68 77 T T T 69 69 78 F IF or I Very unusual residue LOOP H2 1/9A H69 IM But F in 7E3 Vernierzone 70 70 79 S S S 71 71 80 K* R K* or R canonical H2 class 1(16) RKVILOOP H2 1/9A H71 RKV Vernier zone 72 72 81 D D D 73 73 82 T N N Vernierzone 74 74 83 S S S 75 75 84 N K K 76 76 85 N N N 77 77 86 Q T T 78 7887 V L V or L LOOP H1 3/12A H78 FV Vernier zone 79 89 88 F Y Y 80 80 89L L L LOOP H1 3/12A H80 IL 81 81 90 K Q Q 82 82 91 I M M    82A  ^( 82A) 92 A N N   82B  ^( 82B) 93 S S S   82C  ^( 82C) 94 V L L 83 8395 D R R 84 84 96 I A A 85 85 97 A E E 86 86 98 D D D 87 87 99 T T T 8888 100  A A A 89 89 101  T V V 90 90 102  Y Y Y 91 91 103  Y Y Y VH/VLinterface 92 92 104  C C C LOOP H1 3/12A H92 C 93 93 105   I A I or AVH/VL interface CDR3 IMGT Vernier zone FR3- 94 94 106   R* R R*canonical H1 2(6) Vernier zone 95 95 107   M M VH/VL interface CDR3 CDR3CDR3 Kabat Chothia IMGT 96 96 A A 97 97 E E 98 98 G G 99 99 R R 100 100  Y Y 100A 100 A   D D VH/VL interface (+) 100 B 100 B   A A 100 C 100 C  M M 101   101   D D D 102   102   Y Y Y CDR3 CDR3 Kabat ChothiaFR4-103 103  W W W VH/VL interface (+) Vernier zone 104  104  G G G 105 105  Q Q Q 106  106  G G G 107  107  T T T 108  108  S T T 109  109  V VV 110  110  T T T 111  111  V V V 112  112  S S S FR4-113 113  S S S

Legend: The first column (Kabat numbering) gives the residue numberaccording to Kabat et al. (1991); The second column (Chothia numbering)gives the residue number according to Chothia; The third column (IMGTnumbering) gives the IMGT unique Lefranc numbering. The fourth column(mouse 7E3 VH) gives the amino acid sequence of the V_(H) region ofmouse 7E3 anti-TLR4 MD2 antibody used as donor sequence forCDR-grafting; The fifth column (Human Germline IGHV3-66) gives thesequence amino acid of the human germline immunoglobulin heavy variable2-26.used as acceptor sequence for CDR-grafting. The sixth column(Reshaped version 3-66 7E3 VH) gives the amino acid sequence of thereshaped human 7E3 V_(H) region. The mouse amino acid residues which arekept in the humanized version are in yellow.

The positions of framework segments (FR1, FR2, FR3, and FR4) and thecomplementarity—determining regions (CDR1, CDR2, and CDR3) are shown incolumn one. (*) indicates parts of main canonical structure for the CDRloops as defined by Chothia et al. (1989). Bolded entries, notunderlined represent positions in FRs and CDRs where the human and mouseamino acid residues are identical. Italicized entries representpositions in FRs where the human residue differs from the analogousmouse residue number. Underlined entries (bolded or not bolded)represent positions in FRs and CDRs where the human amino acid residuewas replaced by the corresponding mouse residue. Boxed entries representhuman residues conserved in the reshaped human version. TABLE 8Humanized Heavy chain 2-70 Kabat Chothia IGMT Human Human Number-Number- Number- Mouse Germline Version ing ing ing 7E3 VH IGHV2-70 2-707E3 VH Comments FR1-1 FR1-1 FR1-1 Q Q

Vernier zone 2 2 2 V V

3 3 3 T T

4 4 4 L L

5 5 5 K R

6 6 6 E E

7 7 7 S S

8 8 8 G G

9 9 9 P P

10 10 11 G A

11 11 12 I L

12 12 13 L V

13 13 14 Q K

14 14 15 P P

15 15 16 S T

16 16 17 Q Q

17 17 18 T T

18 18 19 L L

19 19 20 S T

20 20 21 L L

LOOP H1 3/12A H20 L 21 21 22 T T

22 22 23 C C

LOOP H1 3/12A H22 C Conserved amino acid 23 23 24 S T

Surface residue 24 24 25 F* F*

canonical H1 2(6) LOOP H1 3/12A H24 VF 25 FR1-25 FR1-26 S S

26

G* G*

canonical H1 2(6) LOOP H1 3/12A H26 G 27

F* F*

canonical H1 2(6) Vernier zone 28

S S

LOOP H1 3/12 A H28 S Vernier zone 29

L* L*

canonical H1 2(6) LOOP H1 2/11 A H29 IL Vernier zone FR1-30

T S

Vernier zone 31 CDR1 Kabat

T T

32

Y S

LOOP H1 2/11A H31A D but G in 15C1 33

N G

34

I M

35 33

G* C*

canonical H1 2(6) LOOP H1 2/11A H34 WV VH/VL interface

34 39 V V

35 40 G S

FR2-36 36 41 W W

LOOP H1 2/11A H36 W Conserved amino acid 37 37 42 I I

VH/VL interface 38 38 43 R R

39 39 44 Q Q

VH/VL interface 40 40 45 P P

41 41 46 S P

42 42 47 G G

43 43 48 K K

44 44 49 G A

45 45 50 L L

VH/VL interface (+) 46 46 51 E E

47 47 52 W W

LOOP H2 1/9A H47 WY VH/VL interface Vernier zone 48 48 53 L L

LOOP H1 3/12A H48 ML Vernier zone FR2-49 49 54 A A

Vernier zone

50 55 H L

Vernier zone

51

I I

LOOP H2 1/9A H51 IMV

W D

W W

LOOP H1 3/12A H53 YW

N D

D* D*

canonical H2 class 1 (16) GD LOOP H2 1/9A H55 G But D in 7E3

N D

57

I K

58 66 Y Y

59 67 Y Y

LOOP H2 1/9A H59 YL

60 68 N S

61 69 T T

62 70 V S

63 71 L L

64 72 K K

65 74 S T

FR3-66 66 75 R R

67 67 76 L L

Verbier zone Close to CDRs 68 68 77 T T

69 69 78 F I

Very unusual residue LOOP H2 1/9A H69 IM But F in 7E3 Vernier zone 70 7079 S S

71 71 80 K* K*

canonical H2 class 1(16) RKVI LOOP H2 1/9A H71 RKV Vernier zone 72 72281 D D

73 73 82 T T

Vernier zone 74 74 83 S S

75 75 84 N K

76 76 85 N N

77 77 86 Q Q

78 78 87 V V

LOOP H1 3/12A H78 FV Vernier zone 79 89 88 F V

80 80 89 L L

LOOP H1 3/12A H80 IL Conserved amino acid 81 81 90 K T

82 82 91 I M

82A 82A 92 A T

82B 82B 93 S N

82C 82C 94 V M

83 83 95 D D

84 84 96 I P

85 85 97 A V

86 86 98 D D

87 87 99 T T

88 88 100 A A

89 89 101 T T

90 90 102 Y Y

91 91 103 Y Y

VH/VL interface 92 92 104 C C

LOOP H1 3/12A H92 C Conserved amino acid 93 93

I A

VH/VL interface Vernier zone FR3-94 94

R* R*

canonical H1 2(6) Vernier zone

I

VH/VL interface

VH/VL interface (+)

FR4-103 103 W

VH/VL interface (+) Vernier zone 104 104 G

105 105 Q

106 106 G

107 107 T

108 108 S

109 109 V

110 110 T

111 111 V

112 112 S

FR4-113 113 S

Legend: The first column (Kabat numbering) gives the residue numberaccording to Kabat et al. (1991); the second column (Chothia numbering)gives the residue number according to Chothia; the third column (IMGTnumbering) gives the IMGT unique Lefranc numbering. The fourth column(mouse 7E3 VH) gives the amino acid sequence of the V_(H) region ofmouse 7E3 anti-TLR4 MD2 antibody used as donor sequence forCDR-grafting; The fifth column (Human Germline IGHV2-70) gives thesequence amino acid of the human germline immunoglobulin heavy variable2-70.used as acceptor sequence for CDR-grafting. The sixth column(Humanized version 2-70 7E3 VH) gives the amino acid sequence of thehumanized version of 7E3 V_(H) region. The mouse amino acid residueswhich are kept in the humanized version are in yellow.

The positions of framework segments (FR1, FR2, FR3, and FR4) and thecomplementarity- determining regions (CDR1, CDR2, and CDR3) are shown incolumn one. (*) indicates parts of main canonical structure for the CDRloops as defined by Chothia et al. (1989). Bolded entries, notunderlined, represent positions in FRs and CDRs where the human andmouse amino acid residues are identical. Italicized entries representpositions in FRs where the human residue differs from the analogousmouse residue number. Underlined entries (bolded or not bolded)represent positions in FRs and CDRs where the human amino acid residuewas replaced by the corresponding mouse residue. Boxed entries representhuman residues conserved in the reshaped human version.

Table 9 presents alignments of the amino acid sequences that were usedto design the humanized 15C1 V_(L) region: TABLE 9 7E3 humanized Lightchain L19 Human Germline FR Mouse L19 Reshaped or 7E3 IGKV1-12 humanComments Kabat Chothia CDR Light IGKV1D-12 7E3 L19 Chothia canonicaldefinitions 1 1 FR1 A D

2 2 | I I

L1 class 2/11A (11) L2 I L3 1/9A (9) L2 ILV 3 3 | Q Q

L3 1/9A (9) L3 VQLE 4 4 | M M

L1 class 2/11A (11) L4 ML L3 1/9A (9) L4 ML 5 5 | T T

6 6 | Q Q

7 7 | S S

8 8 | T P

9 9 | S S

10 10 | S S

11 11 | L V

12 12 | S S

13 13 | A A

14 14 | S S

15 15 | L V

16 16 | G G

17 17 | D D

18 18 | R R

19 19 | V V

20 20 | T T

21 21 | I I

22 22 | N T

23 23 FR1 C C

L1 class 2/11A (11) L23 C

R R

25 25 | A A

L1 class 2/11A (11) L25 A 26 26 | S S

L1 class 2/11A (11) L26 S 27 27 | Q Q

28 28 |

G

L1 class 2/11A (11) L28 NSDE L3 1/9A (9) L28 SNDTE 29 29 | I I

L1 class 2/11A (11) L29 IV 30 30 |

S

L3 1/9A (9) L30 DLYVISNFHGT 31 31 |

S

L3 1/9A (9) L31 SNTKG 32 32 |

W

L3 1/9A (9) L32 FYNAHSR 33 33 | L L

L1 class 2/11A (11) L33 LV L3 1/9A (9) L33 MLVIF

A

L1 class 2/11A (11) L34 AGNSHVF 35 35 FR2 W W

L1 class 2/11A (11) L35 W 36 36 | Y Y

L1 class 2/11A (11) L36 YLF VL/VH interface 37 37 | Q Q

38 38 | Q Q

VL/VH inter 39 39 | K K

40 40 | P P

41 41 | D G

42 42 | G K

43 43 | T A

44 44 | V P

VL/VH interface 45 45 | R K

46 46 | L L

L1 class 2/11A (11) L46 LRV VL/VH interface + 47 47 | L L

Vernier zone 48 48 | I I

L2 class 1/7A (7) IV 49 49 FR2 Y Y

L1 class 2/11A (11) L49 YHFK

A

51 51 |

A

L1 class 2/11A (11) L51 ATGV 52 52 | S S

53 53 | K S

54 54 | L L

55 55 |

Q

S S

57 57 FR3 G G

58 58 | A V

59 59 | P P

60 60 | S S

61 61 | R R

62 62 | F F

63 63 | S S

64 64 | G G

L2 class 1/7A (7) G 65 65 | R S

66 66 | G G

Vernier zone 67 67 | S S

68 68 | G G

Vernier zone 69 69 | T T

Vernier zone 70 70 | D D

71 71 | Y F

L1 class 2/11A (11) L71 YF 72 72 | S T

73 73 | L L

74 74 | T T

75 75 | I I

76 76 | S S

77 77 | N S

78 78 | L L

79 79 | E Q

80 80 | Q P

81 81 | E E

82 82 | D D

83 83 | I F

84 84 | A A

85 85 | T T

86 86 | Y Y

87 87 | F Y

VL/VH interface 88 88 FR3 C C

L3 1/9A (9) L88 C

Q Q

L3 1/9A (9) L89 QSGFL VL/VH inter 90 90 | Q Q

L1 class 2/11A (11) L90 HQ L3 1/9A (9) L90 QNH 91 91 |

A

L3 1/9A (9) L91 NFGSRDHTYV VL/VH inter 92 92 | N N

L3 1/9A (9) L92 NYWTSRQHAD 93 93 |

S

L1 class 2/11A (11) L93 GSNTREA L3 1/9A (9) L93 ENGHTSRA 94 94 | F F

L3 1/9A (9) L94 DYTVLHNIWPS BUT L94 F 95 95 | P P

L3 1/9A (9) L95 P 96 96 | W

L3 1/9A (9) L96 PLYRIWF VL/VH inter+

T

L3 1/9A (9) L97 T 98 98 FR4 F

L3 1/9A (9) L98 F VL/VH inter+ 99 99 | G

100 100 | G

101 101 | G

102 102 | T

103 103 | K

104 104 | L

105 105 | E

106 106 | I

107 107 FR4 K

Legend: The first column (Kabat) gives the residue number according toKabat et al. (1991); The second column (Chothia) gives the residuenumber according to Chothia; The third column (FR or CDR) is convenientto identify the framework segments (FR1, FR2, FR3, and FR4) and thecomplementarity-determining segments (CDR1, CDR2, and CDR3) with thethree CDRs separating the four FRs; The fourth column (mouse 7E3 Light)gives the amino acid sequence of the V_(L) region of mouse 7E3 anti-TLR4MD2 antibody; The fifth column (Human Germline L19 IGKV1-12 IGKV1D-12)gives the sequence amino acid of the human germline Kappa light chainL19 IGKV1-12 IGKV1D-12. The sixth column (Reshaped human 7E3) gives theamino acid sequences of humanized 7E3 light chain.

As used in Table 9, (*) represents part of main canonical structure forthe CDR loops as defined by Chothia et al. (1989). Bolded entries, notunderlined, represent positions in FRs and CDRs where the human andmouse amino acid residues are identical. Italicized entries representpositions in FRs where the human residue differs from the analogousmouse residue number. Underlined entries (bolded or not bolded)represent positions in FRs and CDRs where the human amino acid residuewas replaced by the corresponding mouse residue. Boxed entries representhuman germline gene residues

Example 36 hu18H10 Binds hTLR4 hMD2 Expressed on CHO Cells

In order to demonstrate the ability of the hu18H10 humanized monoclonalantibody to bind to the human TLR4/MD-2 complex, flow cytometryexperiments (as described above) were performed using chimeric 18H10 asa positive control. FIG. 39 shows that hu18H10 bound TLR4/MD-2 in amanner very similar to that of the 18H10 chimeric antibody describedabove.

Example 37 hu7E3 Humanized Monoclonal Antibodies Bind hTLR4 hMD2Expressed on CHO Cells

In order to demonstrate the ability of the hu7E3 humanized monoclonalantibodies to bind to the human TLR4/MD-2 complex, flow cytometryexperiments (as described above) were performed using chimeric 7E3 as apositive control. The hu7E3 antibodies tested included a hu7E3 antibodythat includes VH 2-70 shown in SEQ ID NO:51 and the V_(L) L19 shown inSEQ ID NO:53 (“7E3 2-70/L19”) and a hu7E3 antibody that includes V_(H)3-66 shown in SEQ ID NO:52 and the V_(L) L19 shown in SEQ ID NO:53 (“7E33-66/L19”) FIG. 40 shows that hu7E3 MAbs bound TLR4/MD-2 in a mannersimilar to that of the 7E3 chimeric antibody described above.

Example 38 hu15C1 Humanized Monoclonal Antibodies Bind hTLR4 hMD2Expressed on CHO Cells

In order to demonstrate the ability of the hu7E3 humanized monoclonalantibodies to bind to the human TLR4/MD-2 complex, flow cytometryexperiments (as described above) were performed using chimeric 15C1 as apositive control. The hu15C1 antibodies tested included the hu15C1antibody that includes V_(H) 4-28 shown in SEQ ID NO:45 and the V_(L)A26 shown in SEQ ID NO:48 (“15C1 4-28/A26”) and variants thereof inwhich residues at certain positions (QC ##, Kabat numbering) have beenreplaced by the corresponding amino acids in the given human germline(“15C1 4-28 QC 30/A26”; “15C1 4-28 QC 48/A26”; “15C1 4-28 QC 67/A26” and“15C1 4-28 QC 69/A26”, see Table 1). Other hu15C1 antibodies testedinclude a hu15C1 antibody that includes V_(H) 3-66 shown in SEQ ID NO:46and the V_(L) L6 shown in SEQ ID NO:47 (“15C1 3-66/L6”) and a hu15C1antibody that includes V_(H) 3-66 shown in SEQ ID NO:46 and the V_(L)A26 shown in SEQ ID NO:48 (“15C1 3-66/L6”). FIGS. 41 and 42 demonstratethat the hu15C1 antibodies MAbs bound TLR4/MD-2 in a manner similar tothat of the 15C1 chimeric antibody described above.

Example 39 TLR4 and MD2 Epitope Mapping Studies

hu15C1, hu7E3 and hu18H10 are three monoclonal antibodies (MAbs) showingspecificity for the human TLR4/MD-2 receptor complex. This receptorcomplex is activated by lipopolysaccharide (LPS) the major component ofthe outer membrane of gram-negative bacteria. All three MAbs are capableof inhibiting receptor activation and subsequent intracellular signalingvia LPS, but interestingly, all three have distinct specificities.hu15C1 binds TLR4 independently of the presence of MD-2. hu7E3 binds toTLR4, but binding is greatly enhanced by the presence of MD-2,suggesting that the presence of the latter causes a conformationalchange in TLR4 exposing an epitope bound by hu7E3. hu18H10 binds toMD-2, but requires the presence of TLR4, as the MAb does not bindsoluble forms of MD-2.

The aim of this study was to identify small regions and individual aminoacids of both the TLR4 and MD-2 sequences important for the binding ofhu15C1, hu7E3 and hu18H10. The amino acid sequence of human TLR4(GenBank Accession No. O00206) is shown in FIG. 43. The amino acidsequence of Human MD2 (GenBank Accession No. Q9Y6Y9) is shown in FIG.33B.

As none of the hu15C1, hu7E3 and hu18H10 MAbs demonstratecross-reactivity to the mouse TLR4/MD-2 receptor complex, a strategyutilizing mouse-human hybrids, whereby segments of the human TLR4 andMD-2 proteins were replaced by the equivalent mouse segments was used toidentify defined linear regions of human TLR4 and MD-2 essential for thebinding of the MAbs. Furthermore, these regions were mutated at aminoacid residues differing between the human and mouse sequences in orderto identify individual amino acids essential for the binding of theseMAbs.

Generation of Mouse-Human Hybrid TLR4 Mutants.

To generate the mouse-human-human-human (MHHH), human TLR4, cloned intothe mammalian expression vector pCDNA3.1(−)hygro (Invitrogen) wasmodified by introducing a novel HpaI site and destroying an existingHpaI site by site-directed mutagenesis with the followingoligonucleotides: 5′ GACCATTGAAGAATTCCGGTTCTCTTGCTCTCCTCG 3′ (SEQ IDNO:55); 5′ CGAGGTAGTAGTCTAAGTATGTTAACCGGAATTCTTCAATGGTC 3′ (SEQ IDNO:56) (introduction of novel HpaI site) and 5′ GGCAACATTTAGAATTAGTCAACTGTAAATTTGGACAG 3′ (SEQ ID NO:57); 5′ CTGTCCAAATTTACAGTTGACTAATTCTAAATGTTGCC 3′ (SEQ ID NO:58) (destruction of existing HpaIsite). Site-directed mutagenesis was performed using the QuikChange™ kit(Stratagene) following the manufacturer's instructions. The N-terminalregion of mouse TLR4 was amplified by PCR using the followingoligonucleotides: 5′ ATTTGTATAGTTAACCTGAACTCATC 3′ (SEQ ID NO:59) and 5′GGGGCGGCCGCGGGAAGCTTG AATCCCTGCATAG 3′ (SEQ ID NO:60). This mouse DNAfragment replaced the corresponding human DNA fragment in theHpaI-mutated human TLR4 vector (above) by cloning at the unique NotI andHpaI restriction sites.

To generate HHHM, the C-terminal region of mouse TLR4 was amplified byPCR using the following oligonucleotides: 5′ GGGGATATCTTTGCAAACACAACAAACTTGAC 3′ (SEQ ID NO:61) and 5′ GGGCTCGAGCTTGTACATATAACAG GTAG 3′(SEQ ID NO:62). This mouse DNA fragment replaced the corresponding humanDNA fragment in the human TLR4 vector by cloning at the unique EcoRV andXhoI restriction sites.

To generate MMHH, the MHHH construct was modified by site-directedmutagenesis (as above) to introduce a unique AgeI restriction site intothe TLR4 sequence using the following oligonucleotides: 5′GCTTTTTCAGAAGTTGATCTACCGGTCCTTG AGTTTCTAGATCTCAGT 3′ (SEQ ID NO:63) and5′ ACTGAGATCTAG AAACTCAAGGACCGGTAGATCAACTTCTGAAAAAGC 3′ (SEQ ID NO:64).In parallel, an internal region of mouse TLR4 was amplified by PCR usingthe following oligonucleotides: 5′ CATTGATGAGTTCAGGTTAAC 3′ (SEQ IDNO:65) and 5′ ATGCACCGGTAGGGCCACTTTTTTAAAACTG 3′ (SEQ ID NO:66). Thismouse DNA fragment replaced the corresponding human DNA fragment in theAgeI-mutated MHHH vector (above) by cloning at the unique HpaI and AgeIrestriction sites.

To generate the mouse-human-mouse-human (MHMH) hybrid, an internalregion of mouse TLR4 was amplified by PCR using the followingoligonucleotides: 5′ ATGCACCGGTTCTCAGCTATCTAGATCTTAG 3′ (SEQ ID NO:67)and 5′ ATGCGATATCTGAAAGGGTGTTGTCTTTGAAAG 3′ (SEQ ID NO:68). This mouseDNA fragment replaced the corresponding human DNA fragment in theAgeI-mutated MHHH vector (above) by cloning at the unique AgeI and EcoRVrestriction sites.

To generate MMHHa, an internal region of human TLR4 was amplified by PCRusing the following oligonucleotides: 5′ CCGTTAACATATACAAATGATTTTTCAGATGATATTGTTAAGTTCCATTGCTTGGCGAATGTTTCTGCAATGTCTCTGGCAGGTGTGACTATTGAAAGGGTAAAAG 3′ (SEQ ID NO:69) and 5′ CCACCGGTAGATCAACTTCTGAAAAAGC 3′ (SEQ ID NO:70). This DNA fragment replaced thecorresponding human DNA fragment in the AgeI-mutated MHHH vector (above)by cloning at the unique HpaI and AgeI restriction sites.

To generate MMHHb, an internal region of human TLR4 was amplified by PCRusing the following oligonucleotides: 5′ CCGTTAACATACTTAGACTACTA C 3′(SEQ ID NO:71) and 5′ GATATCTGAAAGGGTGTTGTCTTTGAAAGAATTGCCAGCCATTTTTAATGTGTTGAGACTGGTCAAGCCAAGAAATATACCATCGAAGTCAATTTTGGTGTTAGTATGAGAAATGTCAAG 3′ (SEQ ID NO:72). This DNA fragment replacedthe corresponding human DNA fragment in the AgeI-mutated MHHH vector(above) by cloning at the unique Hpal and AgeI restriction sites.

Generation of Mouse-Human Hybrid MD-2 Mutants.

Firstly, human MD-2, cloned into the mammalian expression vectorpCDNA3.1(−) (Invitrogen) was modified by site-directed mutagenesis (asabove) to introduce a novel AflII restriction site with the followingoligonucleotides: 5′ CTCTTTTTGCAGAGCTCTTAAGGGAGAGACTGTGAA 3′ (SEQ IDNO:73) and 5′ TTCACAGTCTCTCCCTTAAGAGCTCTGCAAAAAGAG 3′ (SEQ ID NO:74).

In order to generate MHH, the N-terminal region of mouse MD-2 wasamplified by PCR using the following oligonucleotides: 5′GGAAGCTTAACCACCATG TTGCC 3′ and 5′ CCGGATCCCCTCAGTCTTATGC 3′ (SEQ IDNO:75). This mouse DNA fragment replaced the corresponding human DNAfragment in the AflII-mutated human MD-2 vector (above) by cloning atthe unique HindIII and BamHI restriction sites.

In order to generate HMH, an internal region of mouse MD-2 was amplifiedby PCR using the following oligonucleotides: 5′ CCGGATCCAATGGATTTGTGCATG 3′ (SEQ ID NO:76) and 5′ GGCTTAAGAGCTCTGCAAAAAGAATAGTC 3′ (SEQ IDNO:77). This mouse DNA fragment replaced the corresponding human DNAfragment in the AflII-mutated human MD-2 vector (above) by cloning atthe unique BamHI and AflII restriction sites.

In order to generate HHM, the C-terminal region of mouse MD-2 wasamplified by PCR using the following oligonucleotides: 5′GGCTTAAGGGAGAGACTG TGAATACATC 3′ (SEQ ID NO:78) and 5′CCGCTAGCATTGACATCACGGC 3′ (SEQ ID NO:79). This mouse DNA fragmentreplaced the corresponding human DNA fragment in the AflII-mutated humanMD-2 vector (above) by cloning at the unique AflII and NheI restrictionsites.

Generation of Human TLR4 and MD-2 Alanine Scanning Mutants.

All mutants were generated by site-directed mutagenesis using theQuikChangem kit (Stratagene) as above. DNA oligonucleotides housing theappropriate mismatch mutations were used with either human TLR4 inpCDNA3.1(−)hygro or human MD-2 in pCDNA3.1(−) as appropriate.Introduction of the desired mutations was verified by DNA sequencing.

Transient Transfection of HEK 293 Cells.

HEK 293 cells, expressing both the large T and EBNA antigens to allowfor episomal plasmid replication, were plated in 1 ml culture medium at1×10⁵ cells/well in 24 well culture plates. The following day, cellswere transfected with 1 μg DNA/well (0.5 μg+0.5 μg of each plasmid forco-transfections) using 1.5 μl/well Fugene6™ transfection reagent(Roche), following the manufacture's guidelines. Cells were analyzed48-72 hours post-transfection.

Flow Cytometry.

Binding of MAb to the surface of TLR4/MD-2 transfected HEK 293 cells wasmeasured by flow cytometry. 1×10⁵ cells were incubated in 96 wellV-bottom plates with the appropriate MAb at a final concentration of 10μg/ml in a volume of 50-100 μl in FACS buffer (1×PBS, 100 μg/ml BSA,0.05% NaN₃). Following a 30 minute incubation at 4° C., cells werepelleted, washed once with 200 μl FACS buffer, repelleted andresuspended with allophycocyanin (APC) conjugated secondary antibody(Molecular Probes) at a 1:250 dilution in FACS buffer. Following a 30minute incubation at 4° C., cells were washed once in 200 μl FACSbuffer, fixed in 1% paraformaldehyde, 1×PBS and analyzed forfluorescence using a FACScalibur (Becton Dickenson) in the FL-4 channel.

hu15C1 and hu7E3 Bind to an 87 Amino Acid Internal Region of TLR4.

Four mouse-human hybrid mutants of TLR4 were generated in order todetermine the precise region of TLR4 responsible for binding to hu15C1and hu7E3. Transient transfection of HEK 293 cells allowed presentationof either wild type (wt) or mutated forms of TLR4 along with wt MD-2 onthe cell surface. FACS analysis (FIGS. 34 a and b) revealed that thecomplex was correctly expressed in three of the four cases (as shown byc-myc and FLAG staining). TLR4 mutant version MHMH was poorly expressionon the cell surface and did not support interaction with MD-2,suggesting that the protein was not conformationally correct. Thisobservation meant that results of binding with hu15C1 and hu7E3 couldnot be taken into account. Whilst versions MHHH and HHHM bound bothhu15C1 and hu7E3 well, MMHH was negative for binding of both antibodies,suggesting that an 87 amino acid internal region of TLR4 (highlighted inFIG. 34 a) is essential for interaction between TLR4 and either hu15C1or hu7E3.

In order to determine in more detail the residues important for hu15C1and hu7E3 binding, two additional mutants were generated whereby eitherthe first 30 amino acids (MMHHa) or the last 32 amino acids (MMHHb) ofthis internal region were replaced by the corresponding mouse sequence(FIG. 35 a). FACS analysis (FIGS. 35 a and b) of transfected HEK 293cells revealed that the complex was correctly expressed in both cases(as shown by c-myc and FLAG staining). hu15C1 bound well to MMHHa butshowed no binding to MMHHb, suggesting that residues situated towardsthe C terminus of this internal region are critical for binding.Conversely, hu7E3 bound well to MMHHb but showed no binding to MMHHa,suggesting that residues situated towards the N terminus of thisinternal region are critical for binding.

HTA125 Recognizes an N-Terminal Region of TLR4.

HTA125 is a commercially available non-neutralizing MAb directed againsthuman TLR4 (E-biosciences). HTA125 was tested against the fourmouse-human hybrids and found, in contrast to the neutralizing MAbs 15C1and 7E3, an absence of binding when the N-terminal region of TLR4 waschanges from human to mouse (FIG. 34 a and c).

TLR4 Amino Acid Residues Essential for hu15C1 and hu7E3 Binding.

In order to identify important residues within the 87 amino acid regionof TLR4 identified above, the human sequence was aligned to thecorresponding mouse TLR4 sequence within this region (alignmentsperformed using the b12seq program). Since hu15C1 and hu7E3 do notcross-react with mouse TLR4/MD-2, it was assumed that residues essentialfor MAb binding would not be conserved between the two species. Allnon-conserved residues in the human sequence were mutated to alanine. 20mutant (QC 1 to QC 20) versions were constructed, each one containingtwo or three residues converted to alanines (FIG. 36 a).

Following transient transfection of these mutants along with wt MD-2 inHEK 293 cells, C-myc and hu18H10 MAbs were used to detect the presenceof TLR4 and MD-2 respectively on the cell surface. FACS analysis (FIG.36 b) showed that all TLR4 mutants were expressed at a level well abovebackground. All mutants bound MD-2 well with the exception of QC 6. Inorder to determine the level of binding of hu15C1 and hu7E3 to themutant TLR4, a “normalized” value was obtained by dividing the meanfluorescence intensity (MFI) obtained with the MAb by that obtained withC-myc. This allowed for variation in the level of expression at the cellsurface between the TLR4 mutants. For hu15C1, normalized binding wasseen to be greatly diminished for versions QC 10, QC 15 and QC 20. Forhu7E3, QC 1, QC 2, QC 6 and QC 7 showed greatly reduced hu7E3 binding,although as hu7E3 required the presence of MD-2 for binding, lack ofbinding to QC 6 could simply be explained by the absence of MD-2 on thecell surface (see hu18H10 MFI for QC 6). These results confirm thatresidues important for hu15C1 binding are located at the C terminal endof the 87 amino acid section identified above, whereas residuesimportant for hu15C1 binding are located towards the N terminal end.

hu18H10 Binds to a 39 Amino Acid N-Terminal Region of MD-2.

Three mouse-human hybrid mutants of MD-2 were generated in order todetermine the precise region of the protein responsible for binding tohu18H10. Transient transfection of HEK 293 cells allowed presentation ofeither wild type (wt) or mutated forms of MD-2 along with wt TLR4 on thecell surface. FACS analysis (FIGS. 37 a and b) revealed that the complexwas correctly expressed in all three cases (as shown by hu15C1 and C-mycstaining). Whilst versions HMH and HHM bound both hu18H10 well, MHH wasnegative for binding, suggesting that a 39 amino acid N-terminal regionof MD-2 (highlighted in FIG. 37 a) is essential for interaction betweenMD-2 and hu18H10.

MD-2 Amino Acid Residues Essential for hu18H10 Binding.

In order to identify important residues within the 39 amino acid regionof MD-2 identified above, the human sequence was aligned to thecorresponding mouse MD-2 sequence within this region (alignmentsperformed using the b12seq program). Since hu18H10 does not cross-reactwith mouse TLR4/MD-2, it was assumed that residues essential for MAbbinding would not be conserved between the two species. Therefore,mutate all non-conserved residues in the human sequence were mutated toalanine. 14 mutant (QC 1 to QC 14) versions were constructed, each onecontaining a single residue converted to alanine (FIG. 38 a).

Following transient transfection of these mutants along with wt TLR4 inHEK 293 cells, hu15C1 and anti-6×HIS MAbs were used to detect thepresence of TLR4 and MD-2 respectively on the cell surface. FACSanalysis (FIG. 38 b) showed that all TLR4 mutants were expressed at alevel well above background, with the exception of QC7 which appears tobe poorly expressed or has lost its ability to interact with TLR4 (n.b.TLR4 was well expressed upon co-transfection with QC 7). In order todetermine the level of binding of hu18H10 to the mutated versions MD-2,a “normalized” value was obtained by dividing the mean fluorescenceintensity (MFI) obtained with the hu18H10 by that obtained with C-myc.This allowed for variation in the level of expression at the cellsurface between the MD-2 mutants. For hu18H10, normalized binding wasseen to be greatly diminished for version QC 13. These results confirmthat a residue important for hu18H10 binding is located within the 37amino acid N terminal section of MD-2 identified above.

Example 40 hu18H10 Humanized Monoclonal Antibody Inhibits LPS-InducedIL-6 Production in Human Whole Blood

In order to demonstrate the neutralizing capacity of the hu18H10humanized monoclonal antibody for LPS, the ability of hu18H10 to inhibitLPS dependent IL-6 induction of human whole blood is tested (asdescribed above). The ability of the hu18H10 antibody to inhibit theeffects of LPS on blood leucocytes is compared to that of the 18H10chimeric antibody described above.

Example 41 hu7E3 Humanized Monoclonal Antibody Inhibits LPS-Induced IL-6Production in Human Whole Blood

To demonstrate the neutralizing capacity of hu7E3 humanized monoclonalantibodies for LPS, the ability of the hu7E3 antibody to inhibit LPSdependent IL-6 induction of human whole blood is tested (as describedabove). The ability of the hu7E3 antibody to inhibit the effects of LPSon blood leucocytes is compared to that of the 7E3 chimeric antibodydescribed above.

Example 42 hu15C1 Humanized Monoclonal Antibody Inhibits LPS-InducedIL-6 Production in Human Whole Blood

To demonstrate the neutralizing capacity of hu15C1 humanized monoclonalantibodies for LPS, the ability of the hu15C1 antibody to inhibit LPSdependent IL-6 induction of human whole blood was tested (as describedabove). The ability of the hu15C1 antibody to inhibit the effects of LPSon blood leucocytes was compared to that of the 15C1 chimeric antibodydescribed above (FIG. 44).

Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

1. An antibody that immunospecifically binds a Toll-like receptor 4(TLR4)/MD-2 complex, wherein the antibody binds to an epitope comprisingone or more amino acid residues on human TLR4 between residues 289 and375 of SEQ ID NO:54.
 2. The antibody of claim 1, wherein said antibodyspecifically binds to an epitope that comprises at least residues 328and 329 of SEQ ID NO:54.
 3. The antibody of claim 1, wherein saidantibody specifically binds to an epitope that comprises at leastresidues 349 through 351 of SEQ ID NO:54.
 4. The antibody of claim 1,wherein said antibody specifically binds to an epitope that comprises atleast residues 369 through 371 of SEQ ID NO:54.
 5. The antibody of claim1, wherein said antibody specifically binds to an epitope that comprisesat least residues 328, 329, 349 through 351 and 369 through 371 of SEQID NO:54.
 6. The antibody of claim 1, wherein said antibody specificallybinds to an epitope that comprises at least residues 293 through 295 ofSEQ ID NO:54.
 7. The antibody of claim 1, wherein said antibodyspecifically binds to an epitope that comprises at least residues 296and 297 of SEQ ID NO:54.
 8. The antibody of claim 1, wherein saidantibody specifically binds to an epitope that comprises at leastresidues 319 through 321 of SEQ ID NO:54.
 9. The antibody of claim 1,wherein said antibody specifically binds to an epitope that comprises atleast residues 293 through 295, 296, 297 and 319 through 321 of SEQ IDNO:54.
 10. An antibody that immunospecifically binds a Toll-likereceptor 4 (TLR4)/MD-2 complex, wherein the antibody binds to an epitopeon human MD-2 between residues 19 and 57 of SEQ ID NO:44.
 11. Theantibody of claim 10, wherein said antibody specifically binds to anepitope that comprises at least residues 53 of SEQ ID NO:44.
 12. Ahumanized antibody that immunospecifically binds to a Toll-like receptor4 (TLR4)/MD-2 complex, wherein the antibody comprises a heavy chain withthree complementarity determining regions (CDRs) comprising an aminoacid sequence selected from the group consisting of GGYSWH (SEQ IDNO:23); YIHYSGYTDFNPSLKT (SEQ ID NO:24); KDPSDGFPY (SEQ ID NO:25); DSYIH(SEQ ID NO:3); WTDPENVNSIYDPRFQG (SEQ ID NO:4SEQ ID NO:4), GYNGVYYAMDY(SEQ ID NO:5); TYNIGVG (SEQ ID NO:33); HIWWNDNIYYNTVLKS (SEQ ID NO:34);and MAEGRYDAMDY (SEQ ID NO:35).
 13. The antibody of claim 12, whereinthe antibody further comprises a light chain with three CDRs comprisingan amino acid sequence selected from the group consisting of the aminoacid sequence of RASQSISDHLH (SEQ ID NO:28); YASHAIS (SEQ ID NO:29);QNGHSFPLT (SEQ ID NO:30); SASSSVIYMH (SEQ ID NO:8); RTYNLAS (SEQ IDNO:9); HQWSSFPYT (SEQ ID NO:10); RASQDITNYLN (SEQ ID NO:38); YTSKLHS(SEQ ID NO:39); and QQGNTFPWT (SEQ ID NO:40).
 14. A humanized antibodythat immunospecifically binds to a Toll-like receptor 4 (TLR4)/MD-2complex, wherein the antibody comprises a light chain with three CDRscomprising an amino acid sequence selected from the group consisting ofthe amino acid sequence of RASQSISDHLH (SEQ ID NO:28); YASHAIS (SEQ IDNO:29); QNGHSFPLT (SEQ ID NO:30); SASSSVIYMH (SEQ ID NO:8); RTYNLAS (SEQID NO:9); HQWSSFPYT (SEQ ID NO:10); RASQDITNYLN (SEQ ID NO:38); YTSKLHS(SEQ ID NO:39); and QQGNTFPWT (SEQ ID NO:40).
 15. The antibody of claim14, wherein the antibody further comprises a heavy chain with threecomplementarity determining regions (CDRs) comprising an amino acidsequence selected from the group consisting of GGYSWH (SEQ ID NO:23);YIHYSGYTDFNPSLKT (SEQ ID NO:24); KDPSDGFPY (SEQ ID NO:25); DSYIH (SEQ IDNO:3); WTDPENVNSIYDPRFQG (SEQ ID NO:4SEQ ID NO:4), GYNGVYYAMDY (SEQ IDNO:5); TYNIGVG (SEQ ID NO:33); HIWWNDNIYYNTVLKS (SEQ ID NO:34); andMAEGRYDAMDY (SEQ ID NO:35).
 16. A humanized antibody thatimmunospecifically binds to a Toll-like receptor 4 (TLR4)/MD-2 complex,wherein the antibody comprises a heavy chain variable amino acidsequence selected from the group consisting of SEQ ID NOs: 45, 46, 49,51 and
 52. 17. The antibody of claim 16, wherein said antibody furthercomprises a light chain variable amino acid sequence selected from thegroup consisting of SEQ ID NOs: 47, 48, 50 and
 53. 18. A humanizedantibody that immunospecifically binds to a Toll-like receptor 4(TLR4)/MD-2 complex, wherein the antibody comprises a light chainvariable amino acid sequence selected from the group consisting of SEQID NOs: 47, 48, 50 and
 53. 19. The antibody of claim 18, wherein saidantibody further comprises a heavy chain variable amino acid sequenceselected from the group consisting of SEQ ID NOs: 45, 46, 49, 51 and 52.20. A method of alleviating a symptom of a pathology associated withaberrant TLR4 signaling, the method comprising administering an antibodyof claim 1 to a subject in need thereof in an amount sufficient toalleviate the symptom of the pathology in the subject.
 21. The method ofclaim 20, wherein the subject is a human.
 22. The method of claim 20,wherein the amount of said antibody sufficient to alleviate the symptomof the pathology associated with aberrant TLR4 signaling is an amountsufficient to reduce LPS-induced pro-inflammatory cytokine production.23. The method of claim 20, wherein said pathology is selected from thegroup consisting of sepsis, ventilator-induced lung injury, acuteinflammation, chronic inflammation, autoimmune diseases and disordersinduced by endogenous soluble stress factors.
 24. The method of claim23, wherein said chronic inflammation is associated with an allergiccondition, or asthma.
 25. The method of claim 23, wherein said pathologyis inflammatory bowel disorder or atherosclerosis.
 26. The method ofclaim 23, wherein said disorder induced by endogenous soluble stressfactors is osteoarthritis or rheumatoid arthritis.
 27. The method ofclaim 23, wherein said endogenous soluble stress factor is Hsp60,fibronectin, heparan sulphate, hyaluronan, gp96, β-Defensin-2 orsurfactant protein A.
 28. A pharmaceutical composition comprising anantibody of claim 1 and a carrier.
 29. A kit comprising an antibody ofclaim 1.